Method of making an in situ sustained biodegradable drug delivery implant by filling an artificial tissue cavity

ABSTRACT

This invention discloses methods and composition to form biodegradable polymer implant arrays in the live tissue. Artificial cavities are created in the live tissue by using laser ablation, oscillating needle, microneedle array and other methods. The cavities are then filled with biodegradable polymer solution. The solvent in the polymer solution is dissipated in the tissue to form a biodegradable polymer implant in artificial cavities. The cavities and implants formed are arranged to form of an array of implants. The biodegradable polymer in the cavity can also be loaded with drug to form biodegradable drug delivery array in the live tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/156,949 filed Oct. 10, 2018, which is a continuation-in-part ofInternational Application PCT/US2017/042798 filed Jul. 19, 2017, whichclaims priority to: U.S. Provisional Patent Application No. 62/515,504filed Jun. 5, 2017; U.S. Provisional Patent Application No. 62/466,291filed Mar. 2, 2017; U.S. Provisional Patent Application No. 62/378,662filed on Aug. 23, 2016; and U.S. Provisional Patent Application No.62/363,839 filed on Jul. 19, 2016, each of these applications beingherein incorporated by specific reference in their entirety for allpurposes.

U.S. application Ser. No. 16/156,949 filed Oct. 10, 2018 is also acontinuation-in-part of U.S. patent application Ser. No. 15/704,792filed Sep. 14, 2017 now U.S. Pat. No. 10,123,980, which is acontinuation of U.S. patent application Ser. No. 15/099,456 filed Apr.14, 2016 now U.S. Pat. No. 9,789,073, which is a continuation-in-part ofU.S. patent application Ser. No. 14/736,007 filed Jun. 10, 2015 now U.S.Pat. No. 9,345,777, which is a divisional of U.S. patent applicationSer. No. 14/209,827 filed Mar. 13, 2014 now U.S. Pat. No. 9,072,678,which claims priority to each of U.S. Provisional Patent Application No.61/946,825 filed Mar. 2, 2014; U.S. Provisional Patent Application No.61/934,795 filed Feb. 2, 2014; U.S. Provisional Patent Application No.61/820,449 filed May 7, 2013; and U.S. Provisional Patent ApplicationNo. 61/786,215 filed Mar. 14, 2013, each of these applications beingherein incorporated by specific reference in their entirety for allpurposes.

FIELD OF THE INVENTION

This invention generally relates to compositions, methods and devicesfor drug/live cell delivery as well as their applications. Moreparticularly, the invention relates to compositions, methods and devicesfor local, and/or systemic sustained drug and live cell delivery,wherein such compositions comprise of drug/live cell microarrays thatare made externally or made in situ and delivered in a sustained manner.The drug microarrays may be made from biostable or biodegradable polymerand may also include a colored or fluorescent additive to aid invisualization during the drug delivery. The present invention alsorelates to methods and devices for preparation and delivery of suchcompositions. The invention aims to achieve precise control over thedrug dose in an implanted microarray to achieve systemic or localtherapeutic effect.

BACKGROUND OF THE INVENTION Prior Art

Sustained Drug Delivery Using Microneedle Array

Drug delivery using microneedle array is rapidly emerging as a new areain the pharmaceutical field. Please refer to recent reviews andreferences therein by T.-M. Tuan-Mahmood et al. (European Journal ofPharmaceutical Sciences, volume 50, Page 623-637, 2013) and M. R.Parasiteet et al. (Advanced Drug Delivery Reviews, volume 56, page581-587, 2004 and E. Larraneta et al., Materials Science and EngineeringR, Volume 104 Page 1-32, 2016). Microneedle array based systemsgenerally consist of micron size microprojections or microneedlessupported on one side with a supporting base or a base patch. Theneedles typically range in size from 25 microns to 2000 microns and areusually arranged in an array format. The drug is either coated onto orencapsulated in the microneedle array. The array along with its backingmaterials is pushed on the skin surface where the microneedles penetratethe epidermis and/or dermis tissue and/or muscular tissue. The needlesdeposit the drug inside the skin tissue where it is made available forlocal or systemic therapeutic effect. Microneedle array can be madeusing biodegradable or biostable materials. If made using biostablematerials, the drug is generally coated on the array surface or themicroarray is used to perforate the skin and the perforations are usedto transport the drug solution across the skin barrier. Thebiodegradable array is left inside the skin tissue after insertion. Ineither case, the microneedle array must have sharp edges to enablesmooth insertion inside the tissue with minimum pressure or force. Thesharp edge limits the use of hard/solid materials in making array andsoft materials generally cannot be used. The arrays are usually madeexternally in a pharmaceutical manufacturing environment andsubsequently made available for clinical use. There is a strong need foralternative methods and compositions to make microneedle array for localand systemic drug delivery with superior performance and quality.

Treatment of Anemia

Iron deficiency or anemia associated with lack of iron in the blood isone of the most important health issues in the world today, especially,in the third world countries. Iron deficiency affects cognitivedevelopment of children from infancy through to adolescence and isbelieved to be associated with increased morbidity rates. Irondeficiency is generally managed through oral supplements and this is notconsidered to be very reliable method to manage anemia. Oral therapy notonly has lower bioavailability of iron but also has side effects such asconstipation. It also has compliance issue because patients may notcomplete the prescribed oral dose regimen. Severe iron deficiency can bemanaged via intravenous route but it requires careful monitoring inhospital settings. Clearly there is a need for better methods andcompositions that can be useful in managing iron deficiency.

Treatment of Onychomycosis

Onychomycosis or infection of the nail is generally caused by a fungus.The infected nail becomes thick or may become discolored, yellow orgreen. The infected nail also becomes brittle and flakes off losing itsnormal shape. The infected nail has a gross look and may adverselyaffect the cosmetic appearance visually. The oral treatment ofantifungal drugs can cause potential side effects to many people (A. B.Nair et al., International Journal of Pharmaceutics, volume 375, page22-27, 2013). The local application of antifungal drug in the infectednail has penetration issues in the nail body. The drug cannot reach thenail plate which is in a deep part of the nail anatomy where infectiongenerally resides. There is hence a need for newer compositions andmethods to manage nail infections.

Surgical Pain Management

Several millions of surgeries are conducted throughout the world everyyear. Each surgical intervention is generally associated with a surgicalpain which is sometimes managed by use of opioids and its derivatives.The use of opioids has side effects such as severe constipation and apotential risk of addiction.

Microimplant Array Containing Live Mammalian Cells

Microneedle arrays are known to deliver vaccines and drug solutions.Prior art is silent on use of this technology for delivery oftherapeutic mammalian live cells, especially encapsulated cells. This isprobably due to difficulty in making live mammalian cell containingarrays under the conditions which can be tolerated by cells. Hydrogelswhich are typically used for cells encapsulation are soft (in hydratedformat) and array needles made from soft materials do not havesufficient hardness and strength (in hydrated form) to serve as amaterial for an array needle. In dry form, hydrogels like hyaluronicacid have sufficient strength to be useful as an array material, howevermammalian cells cannot survive in the dehydrated dry form. This isespecially true for islet cells which are known to control glucose levelby secreting insulin on demand. Clearly there is a strong need forcompositions, methods and devices which can enable delivery of mammaliancells in the microimplant array format for therapeutic use.

Devices for Implantation of Microimplants in Array Format Microarraybased implants have attracted lot of attention due to their utility insustained drug delivery and pain free delivery. The use of biodegradableor dissolvable microarray for sustained drug delivery is also known.Microarray based implants known in the prior art must have a sharp edgefor easy tissue penetration at distal end and a backing material atproximal end for pushing the implants. The sharp edge and othermechanical properties are generally considered as an essential propertyfor microneedle implantation and it also limits the use of certainsofter materials for to be useful as implantable arrays. For example,many hydrogels such as hydrogels used in soft contact lens applicationin fully hydrated form are soft and mechanically weak and therefore maynot have sufficient strength to be implanted in the microneedle arrayformat. Hydrogel materials like hyaluronic acid are generally used indry format where they have sufficient strength and hardness to penetratethe tissue. It will hence be useful to provide devices and methods forimplantation wherein soft materials like hydrogel materials can beimplanted in hydrated format without the use of a sharp edge.Devices and Compositions for Delivery of Drugs, Vaccines or BotulinumToxin in a Solid State

Many injectable drugs like vaccines, protein drugs are sold as solidswhich are dissolved in saline or other liquids to form an injectablesolution. The solution is transferred into a syringe in a sterile mannerand then injected subcutaneously/intramuscularly. The injection volumedetermines the amount of drug injected which needs to be carefullycalculated and administered by a trained medical professional staff. Theentire procedure requires many steps such as trained medicalprofessional, preparation of solution under sterile condition, fillingthe solution in a syringe under sterile conditions and injecting adesired volume in the tissue. It will be beneficial to developcompositions and methods that will reduce/eliminate the number of stepsinvolved in injecting a solution and human errors associated with suchdelivery. The use of one or more sterile needles and syringe and theirproper and safe disposal creates additional financial and regulatorycost to the end user. Botox® is a trade name for Botulinum toxin. Botoxis neurotoxic protein produced by the bacterium Clostridium and is soldto treat variety of medical conditions. Botox is sold as a sterilelyophilized powder which is reconstituted with sterile saline. Each vialcontains 50 to 100 units of drug and physician generally dilutes itprior to use with 1-3 ml saline solution. The solution is injected usinga fine needle syringe at treatment area and the solution has arecommended shelf life of 48 hours. If a given treatment procedurerequires only 10-20 units of the drug, there is a potential to wasterest of the drug solution unless the same solution is used on adifferent patient within its required shelf life stability. The entireprocess involves many steps and each step may be prone to human error.Steps like a measuring a sterile saline volume, adding a sterile liquidin vial, filling the syringe with drug solution and the like are handledby trained human personnel. Each human step is prone to error likemeasurement error, sterility compromise etc. It will be a valuablecontribution to the art if some or majority steps in delivering theBotulinum toxin is reduced or eliminated completely. It will be alsovaluable contribution to the art if the wastage due to limited shelflife of Botulinum toxin solution is reduced and eliminated completely.Removable metal microneedle arrays with liquid delivery of Botoxsolution has been explored in the past (B. M. Torres et al., J ControlRelease. volume 165(2), 146-152 (2013)).

Biodegradable Metal Based Drug Delivery Arrays

Biodegradable metal based devices have long history of human use (CXianhua et al. and X. Gu et al. and cited reference therein, citedherein for reference only). Metal offers remarkable combination oftoughness and hardness which is unmatched by other types of materials.However biodegradable metals such as magnesium based alloys generallycannot be used for sustained drug delivery applications. It will bevaluable contribution to the art wherein biodegradable metal basedmicroneedle arrays have been designed and used for sustained drugdelivery applications. This invention discloses biodegradable devices,designs and compositions based on biodegradable metal.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses the need for compositions, methods anddevices for local and systemic sustained drug/cell delivery. Suchcompositions are made in situ in the body tissue in the form ofmicroimplants incorporating one or more of a drug/cells, a biodegradableor biostable polymer and/or a visualization agent. The present inventionis also directed towards methods for synthesizing such drug bearingmicroimplants in situ by using devices incorporating retrievablemicroneedle devices. Also provided are devices/apparatus and methodsthat enable to implant drug/cell containing arrays. The arrays can beprefabricated and then loaded in the inventive devices for therapeuticuse.

Accordingly, there is a need for such compositions, methods and devicesas summarized herein in some detail.

Therefore, a general aspect of the present invention is to providemethods for sustained drug delivery which are effective at local orsystemic level and thereby more efficient and cause minimal sideeffects.

A further aspect of the present invention is to provide methods forin-situ formation of drug/live cell bearing microimplants in the skintissue such that larger surface area is available for sustained drugdelivery.

Yet another aspect of the present invention is to provide methods forcreation of artificial cavities in the skin tissue such that drugbearing microimplants can be disposed within artificial cavities forsustained drug delivery.

A more specific aspect of the present invention is to provide devicescapable of delivering compositions in microarray form in the skin tissuein a customized manner and do not require an external manufacturing setup and reduce the associated costs of manufacturing in a factoryenvironment.

Another aspect of the present invention is to provide microarray basedcompositions that are dissolvable and biodegradable and have therapeuticuse.

A further aspect of the present invention is to provide methods anddevices capable of creating a plurality of microimplants comprising ofdrug bearing compositions within the skin tissue, formed in-situ atpredetermined location and having a predefined shape and surface area.

Yet another aspect of the present invention is to provide method fortreating nail infection. This invention provides methods andcompositions to manage such nail infections.

A further aspect of the present invention is to provide methods andcompositions for delivery of drugs like Botox in solid state. Thisinvention bypasses the solution making steps and injects thecompositions in solid state without forming solution eliminating the useof sterile syringe and needles.

A further aspect of the present invention is to provide a method fortreating anemia.

A still further aspect of the present invention is to providecompositions for local anesthetic effect that can be used for surgicalpain management. In this invention, compositions and methods fortreatment of surgical pain are described. In particular, the inventivecompositions and methods deliver bupivacaine based compositions forsurgical pain management.

Still another aspect of the present invention is to provide method forefficient insulin delivery.

A further aspect of the present invention is to provide a method fortherapeutic cell therapy.

Another aspect of this invention is to form a microimplant array in thelive tissue or prosthesis tissue for cell or sustained drug deliverywherein the implanted microimplant does not need a sharp edge. Thisinvention provides devices and methods of implantation whereinmicroimplant array can be formed from softer materials for sustaineddrug delivery compositions or with live cells and without the need ofsharp edge.

Yet another aspect of the present invention is to provide devices,methods and composition for delivery of Botox and other proteindrugs/vaccines in a painless, safe, and hygienic manner in the solidstate form, thereby improving the treatment efficacy as well aseliminating problems associated with safe disposal of medical wastes,human effort, error and inaccuracy.

A further aspect of the present invention is to provide additionalenhancements and improvements in the process of vaccine deliverymethods, including encoding useful information while imparting thevaccines.

One embodiment of the present invention provides a method for creating adrug delivery composition inside the human or animal body wherein themethod comprises: creating artificial porosity inside the human body orskin tissue; partially or completely filling the cavity with aninjectable composition comprising biodegradable or biostablemicroparticles suspended in a biocompatible fluid carrier. The preferredcompositions comprise visualization agent.

One embodiment of the present invention provides a method for creating adrug delivery composition inside the human or animal body wherein themethod comprises: creating artificial porosity inside the human body orskin tissue; partially or completely filling the cavity with aninjectable composition comprising liquid carrier and bioactive compound.The injectable composition stays substantially in liquid state fortherapeutic effect or until biodegradation process is initiated.

One embodiment of the present invention provides a method for creating adrug delivery composition inside the human or animal body wherein themethod comprises: creating an artificial porosity inside the human bodyor skin tissue; partially or completely filling the cavity withinjectable composition comprising biostable or biodegradable meltedpolymer (melting point 60 degree C. or less) and drug; cooling thecomposition inside the cavity to body temperature to form a solid orsemisolid implant in the cavity.

One embodiment of the present invention provides a method for creating adrug delivery composition inside the human or animal body wherein themethod comprises: creating an artificial porosity inside the human bodyor skin tissue; partially or completely filling the cavity withinjectable composition comprising biostable or biodegradable polymerdissolved in a water miscible biocompatible solvent and drug; dispersingthe solvent in the surrounding tissue and precipitating polymer in thecavity and entrapping the drug.

One embodiment of the present invention provides a method for creating adrug delivery composition inside the human or animal body wherein themethods comprises: creating an artificial porosity inside the human bodyor skin tissue; partially or completely filling the cavity withinjectable composition comprising crosslinkable polymer precursors anddrug/cells; crosslinking the precursors to form crosslinked compositionand entrapping the drug/cells; releasing the drug locally from thecrosslinked composition for systemic or local therapeutic effect.Preferred crosslinked composition is biodegradable.

One embodiment of the present invention provides a method for creating adrug delivery composition inside the human or animal body wherein themethod comprises: creating an artificial porosity inside the human bodyor skin tissue; partially or completely filling the cavity withinjectable composition comprising water insoluble drug solution in awater miscible organic solvent; dispersing the solvent and precipitatingthe drug crystals/solids inside the cavity. The precipitated drugsolids/crystals release the drug by slow dissolution or biodegradationprocess.

One embodiment of the present invention provides a method for creating adrug delivery composition inside the human or animal body wherein themethod comprises: creating an artificial porosity inside the human bodyor skin tissue; completely or partially filling the porosity withinjectable thermoreversible or pH sensitive gelling compositions influid state and drug; gelling the composition using thermoreversibleproperty or gelation due to change in pH and entrapping the drug in thegel; releasing the drug locally from the gelled thermoreversiblecomposition for systemic or local therapeutic effect. Preferredthermoreversible composition is biodegradable.

Another embodiment of this invention provides a method for treating nailinfection, wherein the method comprises: creating an artificial porosityinside the nail body; filling the porosity with an injectablecomposition comprising an antifungal or antimicrobial compound.Optionally applying a nail polish or other cosmetic device/coating overthe implanted nail surface to improve cosmetic appearance.

Another embodiment of this invention provides a method for treating irondeficiency. The method involves following steps: a) provide aninjectable composition comprising iron complex or ferric pyrophosphatedissolved or suspended in a biocompatible liquid; b) injecting thecomposition using oscillating needle or a microneedle array under theskin; c) dissociating the complex in skin tissue to release the iron. Inthis invention, iron deficiency is managed by delivery of iron basedcompositions through the skin using oscillating needle or microneedlearray based iron bearing compositions. Iron based microimplants arraycan be made in situ or may be prefabricated and implanted as describedin this invention.

One embodiment of the present invention provides a method for deliveringsustained release of bupivacaine composition for local anestheticeffect. The method involves following steps: a) provide an injectablecomposition comprising bupivacaine dissolved or suspended in abiodegradable polymer dissolved in a water miscible biocompatiblesolvent; b) injecting the composition using oscillating needle or amicroneedle array under the skin; c) dispersing the solvent in the skintissue and precipitating the polymer under skin and entrapping thebupivacaine; d) releasing the bupivacaine in a sustained manner in thetissue.

Another embodiment of this invention discloses a device wherein thedevice has inner and outer parts. The device is inserted in the bodywith inner part inside the outer part; the inner and outer part areseparated to create a cavity inside the device. The cavity is thenfilled with an injectable composition which conforms to the shape of thecavity and then converted into solid or gel state in situ in the cavity.The inner and outer portions of the device are withdrawn leaving behindthe formed implant.

Another embodiment of this invention discloses an “array in array”device to form a microimplant array in the body. One of the arrays(outer array) has hollow needles whose cavities may be filled with aninjectable composition or a preformed implant with drug or live cells.The other array (inner array) has needles in the same format as outerarray that can be easily inserted inside the hollow cavities of theouter array. The needles in the inner array can mechanically,magnetically or via gas pressure push or hold the microimplant in theouter array. The outer array and inner array are removed from the bodyleaving behind the preformed microimplants or in situ formedmicroimplants in the body.

Another embodiment of this invention discloses an “array in array”device to form microimplant array in the body. One of the arrays hashollow needles whose cavities may be filled with an injectablecomposition or preformed implant with drug or live cells. The otherarray has needles that can be easily inserted inside the hollow needlesof the array and can push in situ formed microimplant or preformedmicroimplant inside the body.

One embodiment of the present invention provides a method for creating amicroimplant array comprising live cells inside the human or animal bodywherein the method comprises: providing a hollow microneedle array;filling the cavities of hollow microneedle array with hydrogelscomprising live cells; inserting the array containing cells inside thebody; pushing/expelling hydrogels with cells out of the hollow cavityinto the body; removing the hollow array from the body leaving behindthe cell based hydrogel array inside the body. The hydrogel used in thearray may be biodegradable or biostable. In this invention,compositions, methods and devices are disclosed which enable implant oflive cells in an array format. Cells like islet cells implanted in anarray format, preferably under the skin, survive and produce insulin ondemand. The array like format creates a controlled isolated environmentfor each cell or group of cells where each cell in the array can getnutrients from the surrounding tissue and provide needed therapeuticcompounds such as insulin on demand. If desired, cells may be immunoisolated by using microencapsulation techniques known in the prior artbefore implantation in the array format. Cells may also be encapsulatedduring the implantation in an array format. This invention providesmethods, devices and compositions to create mammalian cell based arrayin live tissue.

One embodiment of this invention provides a dissolvable microimplantarray based compositions comprising iron salts.

One embodiment of this invention provides a biodegradable microimplantarray based compositions comprising cells for therapeutic use. Thepreferred compositions comprise biodegradable hydrogels with livemammalian cells implanted in the skin or body in an array format.

One embodiment of this invention provides a biodegradable microimplantarray based compositions comprising crosslinked polyethylene glycolbased synthetic biodegradable crosslinked gels. The crosslinked gels aremade by free radical polymerization of biodegradable macromonomers. Thecrosslinked gels also can be made by condensation polymerization byreaction of polyethylene glycol comprising precursors. The PEG basedprecursors with nucleophilic and electrophilic reactive groups having atleast five total reactive groups are reacted to produce crosslinkedgels.

One embodiment of this invention provides a biodegradable hydrogel basedmicroimplant array compositions that are reinforced using biodegradablemicroparticles/microspheres or inorganic or organic water solublebiocompatible salts like sodium chloride.

One embodiment of this invention provides biodegradable microarray basedcompositions comprising polyethylene glycol based degradable polymerssuch as polyethylene glycol-polylactone block copolymers,PEG-polytrimethylene carbonate block copolymers.

Another embodiment of this invention discloses an apparatus for makingmicroimplant array in the tissue. The apparatus has specialized needleswhich can be inserted in the body at desired depth and a cavity is thencreated inside the needle while in the tissue. The cavity can be filledwith the injectable composition which may form in situ implant in theneedle. After implant is formed, the needle can be pulled from thetissue leaving behind the implant.

Another embodiment of this invention discloses a device for makingbiodegradable microimplant array in the tissue. The device hasspecialized biodegradable metal microneedles which are coated or infusedwith biodegradable drug delivery composition and has a flexible backingthat enables insertion of arrays in the skin tissue. This inventiondiscloses biodegradable devices, designs and compositions based onbiodegradable metal. The inventive devices are biodegradable metal basedmicroneedle implantable arrays for sustained drug or live cell delivery.

One embodiment of this invention provides biodegradable microarray basedcompositions comprising Botulinum toxin wherein each array needlecomprises a bulking agent and total Botulinum toxin concentration ineach array needle ranges from 0.01 units to 5 units. In this invention,the use of making Botulinum toxin solution prior to delivery iscompletely eliminated and the drug is delivered in the treatment area inthe solid-state microimplant form where it dissolves in situ in thetissue and provides therapeutic action. The preferred compositions arefluorescent/colored microimplants which deliver the Botulinum toxin as asolid microimplant.

One embodiment of this invention provides injectable compositions forsustained drug delivery comprising biodegradable polymer solution inwater miscible organic solvent and biodegradable inorganic salt/spolymeric/hydrogel microparticles as filler materials. Preferablypolymeric materials are crosslinked.

According to one embodiment of the present invention, an “array inarray” (AIA) device comprises: a base array, a plunger array, andoptionally a spacer lock. The base array further comprises a base arrayplate, having a top surface and a bottom surface, and a plurality ofhollow microneedles provided in an array format provided on the bottomsurface of the base array plate. Optionally, a plurality of guidingposts may be provided on the bottom top surface of the base array plate.The plunger array further comprises a plunger array plate, having a topsurface and a bottom surface, and a plurality of solid microneedlesprovided in an array format on the bottom surface of the plunger arrayplate. Optionally, a plurality of guiding holes may be provided on theplunger array plate. The base array and the plunger array are verticallyaligned and dimensionally characterized, such that the plurality ofsolid microneedles of the plunger array is smoothly inserted in theplurality of the hollow microneedles of the base array.

The foregoing discussion summarizes some of the more pertinent objectsof the present invention. These objects should be construed to be merelyillustrative of some of the more prominent features and applications ofthe invention. Applying or modifying the disclosed invention in adifferent manner can attain many other beneficial results or modifyingthe invention as will be described. Accordingly, referring to thefollowing drawings may have a complete understanding of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The above-mentioned and other features and advantages of this presentdisclosure, and the manner of attaining them, will become more apparentand the present disclosure will be better understood by reference to thefollowing description of embodiments of the present disclosure taken inconjunction with the accompanying drawings, wherein:

FIGS. 1A, 1B and 1C are partial schematic representative diagramsillustrating the comparison of an array formed by a conventionalinjectable in situ gelation drug delivery system as known in prior art(FIGS. 1A and 1B) and microimplant array comprising drugs/cells formedby methods disclosed in this invention (FIG. 1C).

FIG. 2 is a partial schematic representative diagram illustrating amethod for forming drug delivery implants in the tissue wherein theartificial cavities are formed first and then are filled with theinjectable drug delivery compositions.

FIG. 3 is a partial schematic representative diagram illustrating amethod for forming drug delivery implants in the tissue wherein a layerof injectable composition is first applied on the tissue followed byinserting a cavity making device such as microneedle array oroscillating needle through the liquid layer to form cavity and fillingthe cavity with drug delivery compositions.

FIG. 4 is a partial schematic representative diagram illustrating amethod for forming drug delivery implants in the tissue wherein theartificial cavities are formed first using dissolvable microneedle arraywhich are then filled with injectable drug delivery compositions.

FIG. 5 shows a partial schematic representation of the epidermis layerand dermis layers along with hollow coated needle such that the coatingon needle prevents the insertion of tissue inside the cavity duringinsertion.

FIGS. 6A, 6B, 6C and 6D show a partial schematic representation of an“array in array” apparatus for creating microimplant array with drugs orlive cells.

FIG. 7 is a partial schematic representative diagram illustrating amethod for forming drug delivery implants in the tissue wherein theartificial cavities are formed first which are then filled withinjectable drug delivery compositions comprising drug encapsulatedmicroparticles.

FIGS. 8A, 8B, 8C, 8D and 8E shows representative images of cavitiesformed in tissue or gelatin gel and then filled with injectablecompositions like biodegradable polymers with drug and/or visualizationagent.

FIG. 9A shows a representative photographic image of iron containingimplant formed inside the tissue using methods described in thisinvention.

FIG. 9B1 depicts a photographic image of 10×10 microimplant array madefrom sodium hyaluronate and iron pyrophosphate.

FIG. 9B2 shows a microscopic image of a sharp tip of one of the needlesof array shown in FIG. 9B1.

FIG. 10 shows a drug release profile of moxifloxacin from microimplantsarray formed in the tissue in an embodiment of the present invention.

FIG. 11 shows a drug release profile of moxifloxacin from microimplantsarray formed in the tissue in an alternate embodiment of the presentinvention.

FIG. 12 shows a drug release profile of moxifloxacin from microimplantsarray formed in the tissue in another alternate embodiment of thepresent invention.

FIG. 13 shows a drug release profile of bupivacaine from microimplantsarray formed in the tissue using an oscillating needle device.

FIG. 14 shows a drug release profile of rifampin encapsulatedmicrospheres from microimplants array formed in the tissue using amicroneedle array in accordance with one embodiment of the presentinvention.

FIGS. 15A-15F show illustrative images of microneedle “array in array”working device for in situ casting of microimplant or in situ insertionprefabricated microimplants made in accordance with one embodiment ofthe present invention.

FIGS. 15G-15H show images of the array formed from the devices of FIGS.15A-15F.

FIGS. 16A, 16B and 16C show creation of artificial cavities in an arrayformat in parts of a human nail, and the release profile of theantifungal drug from the biodegradable array made in accordance with oneembodiment of the present invention.

FIG. 17 shows bupivacaine base release profile of PLGA coatedbiodegradable tissue or tissue based suture threads.

FIG. 18 shows partial schematic representation of microneedle arraycomprising the islets of Langerhans implanted in a skin tissue inaccordance with one embodiment of the present invention.

FIGS. 19A and 19B show exemplary photographic images of microimplantarrays created using methods and devices according to the presentinvention. FIG. 19A shows image of 4 by 4 microimplant array made insheep skin, where the array is an exemplary synthetic biodegradablecrosslinked hydrogel (white colored, opaque) containing magnesiumcarbonate encapsulated microparticles as a visualization agent. FIG. 19Bshows image of 10 by 10 microimplant array made in sheep skin, where thearray is an exemplary liquid carrier vitamin E acetate containing teastained magnesium carbonate (red colored) added as a visualizationagent, and the array is liquid at ambient/body temperature.

FIG. 20 shows a release profile of iron from the treated tissue, infusedwith ferric pyrophosphate and PLGA polymer and control sample is infusedwith PLGA polymer only.

FIG. 21A shows partial schematic representation of a method for makingin situ implant in the human or animal body comprising biodegradablefillers. FIG. 21A shows steps involved in making the implant withfiller.

FIG. 21B shows release profile of bupivacaine hydrochloride from the insitu made PLGA array implant with and without magnesium carbonate asexemplary filler.

FIGS. 22A, 22B, 22C and 22D show exemplary photographic images ofmicroimplant arrays created according to present invention. FIG. 22Ashows a microneedle array containing 20 microneedles used to create 20micro cavities per insertion in the tissue. FIG. 22B shows 33 MP hollowmicroneedle array with 3 by 3 hollow microneedles attached to a syringecontaining injectable composition (PDLG 5002 biodegradable polymersolution in DMSO with methylene blue as a visualization agent). FIG. 22Cshows a 3 by 3 array of fluorescent biodegradable cylindrical rods (100microns diameter and 1000 microns height prepared by slicing 100 microndiameter fluorescent thread) and inserted in the tissue to formmicroimplant array. FIG. 22D shows image of 4 by 4 microimplant arraymade in sheep skin, where the array is an exemplary syntheticbiodegradable thermosensitive polymer hydrogel containing rifampinencapsulated microspheres (red colored) for sustained drug delivery aswell as visualization agent.

FIGS. 23A-23C shows schematic representation of use of expandable arrayneedle in forming drug delivery microimplant array.

FIG. 24 depicts a partial schematic representation of another version of“array in array” device in an alternate embodiment.

FIG. 25 shows a partial schematic representation method to make base orplunger array according to present invention.

FIG. 26A to 26E show partial schematic representation of variousconfigurations of degradable metal based, preferably magnesium alloybased, microneedles arrays that can be useful in sustained drug deliveryapplications.

The figures are not necessarily drawn to scale unless specificallyindicated.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the present disclosure is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thedrawings. The present disclosure is capable of other embodiments and ofbeing practiced or of being carried out in various ways. Also, it is tobe understood that the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting.Exemplary embodiments of the present invention are directed towardscompositions, methods and devices for facilitating local and sustaineddrug/cell delivery.

It is advantageous to define several terms, phrases and acronyms beforedescribing the invention in detail. It should be appreciated that thefollowing terms are used throughout this application. Where thedefinition of terms departs from the commonly used meaning of the term,applicant intends to utilize the definitions provided below, unlessspecifically indicated. The following definitions are provided toillustrate the terminology used in the present invention. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one who is skilled in the art. Allscientific literature and patent citations in this invention areincorporated herein for reference use only.

“Crosslinked material” is meant to denote the formation ofintermolecular or intramolecular covalent bonds in the macromolecule orpolymer. The crosslinked material may be in a highly hydrated state.

A “crosslinking agent” is defined as a compound capable of formingcrosslinked material. For example, glutaraldehyde is generally known inthe art as crosslinking agent for the tissue or with albumin or withcollagen.

“In situ” is meant to denote at a local site, especially within or incontact with living organisms, tissue, skin, organs, or the body.

“Bioprosthesis” is defined to include any prosthesis, which is derivedin whole or in part from animal or other organic tissue includingcultured tissue and which is suitable for human or animal implantation.

The term “tissue/s” incorporates live human or explanted animal tissuefor bioprosthesis used. Generally human organ tissue surface is used inmost cases. The term tissue includes but is not limited to skin tissue,nails, bones, internal organ tissue surfaces such as beating hearttissue surface, arterial tissue surface accessed via catheter based MISsurgical techniques, abdominal tissue surface, peritoneal cavitysurface, internal organ surfaces such as liver, large and smallintestine surface, lung surface and the like. The preferred tissuesurface is a skin tissue surface, membrane like tissue surface likepericardium tissue, bladder tissue and the like and the most preferredtissue is epidermal, dermal tissue or muscular tissue of the human body.The term ‘tissue’ also includes bioprosthesis tissue surface such asheart valve bioprosthesis, tissue based hernia patch, tissue basedsurgical patch, animal tissue based wound dressings and the like.

“Bioactive” refers to one or all of the activities of a compound thatshow pharmacological or biological activity in human or animal body.Such biological activity is preferred to have a therapeutic effect.Substances or compounds that are bioactive are referred to as “drugs” or“bioactive compounds.” The bioactive compounds that can be used include,but are not limited to, antiviral agents; antiinfectives such as, by wayof example, and not limitation, antibiotics; antiviral agents,antifungal agents, antibacterial agents, antipruritics; anticanceragents, antipsychotics; cholesterol- or lipid-reducing agents; cellcycle inhibitors; antiparkinsonism drugs; HMG-CoA inhibitors;antirestenosis agents; antiinflammatory agents; antiasthmatic agents;anthelmintic; immunosuppressives; muscle relaxants; antidiuretic agents;vasodilators; nitric oxide; nitric oxide-releasing compounds;beta-blockers; hormones; antidepressants; decongestants; calcium channelblockers; growth factors such as, by way of example, and not limitation,bone growth factors or bone morphogenic proteins; wound healing agents;analgesics and analgesic combinations; local anesthetic agents;antihistamines; sedatives; angiogenesis-promoting agents;angiogenesis-inhibiting agents; tranquilizers and the like; cellularelements, which can be used for therapeutic use, include, but are notlimited to mammalian cells including stem cells; cellular components orfragments, enzymes, DNA, RNA, and genes may also be included asbioactive components or drugs. Extensive list of bioactive compounds ordrugs that may be used can be found in U.S. Pat. No. 8,067,031 citedherein for reference only.

The terms “Biodegradable” “Bioerodible” and “Bioabsorbable” have thesame meaning unless specified. The terms are meant to denote a materialor substance, that will degrade in a biological environment such ashuman body by either a biologically assisted mechanism, such as anenzyme catalyzed reaction or by a chemical mechanism which can occur ina biological medium, such as hydrolysis or by a dissolution mechanism inwhich the substance dissolves and is removed safely without anydegradation.

“Biostable” is meant to denote a high chemical stability of a compoundin an aqueous environment, which is similar to the environment found inthe human body such as phosphate buffered saline (pH 7.2).

The term “biodegradable polymers” may include polymers or macromoleculeswhich degrade/dissolve safely in the biological environment such as inhuman body. The term applies to polymers that are hydrophobic orhydrophilic. The term is applicable to polymers that are crosslinked ornon-crosslinked. The crosslinking may be done via condensationpolymerization or via free radical polymerization or via ionic bonding.The biodegradable polymers may be random or block or graft copolymers.The biodegradable polymers may be linear, graft, dendramer or branched.The hydrophobic biodegradable polymers include, but are not limited to,polymers, dendramers, copolymers or oligomers of glycolide, dl-lactide,d-lactide, l-lactide, caprolactone, dioxanone and trimethylenecarbonate; degradable polyurethanes; degradable polyurethanes made byblock copolymers of degradable polylactone such as polycaprolactone andpolycarbonate such as poly(hexamethylene carbonate); tyrosine-derivedpolycarbonates, tyrosine-derived polyacrylates; polyamides; polyesters;polypeptides; polyhydroxyacids; polylactic acid; polyglycolic acid;

polyanhydrides; and polylactones. Biodegradable polymers also includepolyhydroxyalkanoates, which are polyesters produced by microorganismsincluding and not limited to poly(3-hydroxybutyrate), 3-hydroxyvalerate,4-hydroxybutarate, 3-hydroxyhexanoate, 3-hydroxyoctanoate.

The term applies to hydrophilic polymers, which include, but are notlimited to, polyethylene glycol-polyhydroxy acid or polyethyleneglycol-polylactone copolymers (PEG-PL copolymers); polyvinylalcohol-co-polylactone copolymers; and derivatives of cellulose;collagen or modified collagen derivatives; gelatin; albumin orcrosslinked albumin; fibrinogen; keratin; starch; hyaluronic acid anddextran.

The term “biostable polymers” include but are not limited to aliphaticand aromatic polyurethanes; polycarbonate polyurethane; polyetherpolyurethane; silicone polyurethane block copolymers; silicone rubbers;polydimethylsiloxane copolymers; polytetrafluoroethylene and otherfluorinated polymers; expanded polytetrafluoroethylene; polyethylene;polyesters, polyethylene terephthalate, polyimides, polypropylene;polyamide; polyamide block copolymers and the like. The polymers must bebiocompatible and suitable for implantation in the human or animal body.

“Sustained release” or “controlled drug delivery” or “long term release”or “deliveries” are phrases used interchangeably herein, to mean longerthan the expected delivery of a bioactive compound from the inventivecomposition. Typically, delivery will be at least for one hour or more,two to six hours or more, and may extend to one day, few days, weeks,months to few years. The long term release can be achieved by any of anumber of known or yet to be discovered or unknown mechanisms.

A “hydrogel” as used herein, refers to a semisolid compositionconstituting a substantial amount of water, and in which polymers,macromolecules or non-polymeric materials or mixtures thereof aredissolved or dispersed. The polymers may be physically or chemicallycrosslinked or not crosslinked.

Polyethylene glycol (PEG) or polyethylene oxide (PEO) refers to the samepolymer, which is made by polymerization of ethylene oxide.

Polypropylene glycol (PPG) or polypropylene oxide (PPO) refers to thesame polymer, which is made by polymerization of propylene oxide.

Polymeric nomenclature used in this patent application such as poly(ethylene glycol) or polyethylene glycol or polyethyleneglycol refer tothe same polymer, unless otherwise stated clearly.

This is also true for all others polymers referred in this patentapplication.

The term “micron” means a length of 1/1000000 of a meter.

The term “micro-implant/s” “microimplant/s” has same meaning.Microimplants are small size implants with an implant volume of 0.05 mlor less.

The term “microimplant array” is defined as group of, two but preferablythree or more microimplants arranged or implanted in symmetrical ornon-symmetrical fashion. A simple symmetric microimplant array may haverows and columns. The microimplants in the array are in close proximitywith each other, such as having a separate distance of range of 10microns to 5 mm.

The term “macromonomer” or “macromer” refers to oligomeric or polymericmaterials capable of undergoing free radical polymerization.

The term “hydrophobic” is defined as a property of materials or polymersor macromolecules having a low degree of water absorption or attraction.

The terms “coloring compositions” include any coloring composition orchemical that is suitable for human or animal implantation and arepreferably approved by FDA for use in implantable medical devices. Thecompounds include but are not limited to: Methylene blue; Eosin Y;Fluorescein sodium; Chromium-cobalt-aluminum oxide; Ferric ammoniumcitrate; Pyrogallol; Logwood extract;1,4-Bis[(2-hydroxy-ethyl)amino]-9,10-anthracenedionebis(2-propenoic)ester copolymers(3; 1,4-Bis[(2-methylphenyl)amino]-9,10-anthracenedione;1,4-Bis[4-(2-methacryloxyethyl) phenylamino] anthraquinone copolymers;Carbazole violet; Chlorophyllin-copper complex, oil soluble;Chromium-cobalt-aluminum oxide; Chromium oxide greens; C.I. Vat Orange1; 2-[[2,5-Diethoxy-4-[(4-methylphenyl)thiol]phenyl]azo]-1,3,5-benzenetriol; 16,23-Dihydrodinaphtho [2,3-a:2′,3′-i]naphth [2′,3′:6,7] indolo [2,3-c] carbazole-5,10,15,17,22,24-hexone;N,N′-(9,10-Dihydro-9,10-dioxo-1,5-anthracenediyl) bis benzamide;7,16-Dichloro-6,15-dihydro-5,9,14,18-anthrazinetetrone;16,17-Dimethoxydinaphtho (1,2,3-cd:3′,2′,1′-lm) perylene-5,10-dione;Poly(hydroxyethyl methacrylate)-dye copolymers: one or more of ReactiveBlack 5; Reactive Blue 21; Reactive Orange 78; Reactive Yellow 15;Reactive Blue No. 19; Reactive Blue No. 4; C.I. Reactive Red 11; C.I.Reactive Yellow 86; C.I. Reactive Blue 163; C.I. Reactive Red 180;4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one;6-Ethoxy-2-(6-ethoxy-3-oxobenzo[b] thien-2(3H)-ylidene)benzo[b]thiophen-3(2H)-one; Phthalocyanine green; Iron oxides; Titaniumdioxide; Vinyl alcohol/methyl methacrylate-dye reaction products; one ormore of: (1) C.I. Reactive Red 180; C.I. Reactive Black 5; C.I. ReactiveOrange 78; C.I. Reactive Yellow 15; C.I. Reactive Blue No. 19; C.I.Reactive Blue 21; Mica-based pearlescent pigments; Disodium1-amino-4-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate (Reactive Blue 69); D&CBlue No. 9; D&C Green No. 5; [Phthalocyaninato(2-)] copper; FD&C BlueNo. 2; D&C Blue No. 6; D&C Green No. 6; D&C Red No. 17; D&C Violet No.2; D&C Yellow No. 10; and the like. Preferred colored compositions arebiodegradable.

The term “minimally invasive surgery” or (MIS) is used herein includes,but is not limited to, surgical techniques such as, by way of example,and not limitation, laparoscopy, thoracoscopy, arthroscopy, intraluminalendoscopy, endovascular techniques, catheter-based cardiac techniques(such as, by way of example, and not limitation, balloon angioplasty),and interventional radiology. The term “hydrophilic” is defined as aproperty of materials or polymers or macromolecules having a strongaffinity for water.

“Polylactic acid” or “poly(lactic acid)” or “poly(lactide)” or PLA isterm used for a polymer which is made from lactide or lactic acid.Similarly, PGA is a term used for polyglycolic acid or polyglycolate.Some synthetic biodegradable polyesters polymers are generally referredto as polylactones or polyhydroxyacids. The terms “PLGA” and “PDLG”refer the same polymer and is a copolymer of PLA and PGA.

The term “oscillating” used in this patent application refers to andfrom motion of a needle along its transversal axis and preferablyperpendicular to the tissue.

The term “polymerizable” denotes the characteristic of molecules thathave the capacity to form additional covalent bonds resulting in monomerand/or monomers interlinking to oligomer or polymer formation, forexample, molecules contain carbon-carbon double bonds of acrylate-typemolecules. Such polymerization is characteristically initiated byfree-radical formation, for example, resulting from photon absorption ofcertain dyes and chemical compounds to ultimately produce free radicals.The term polymerizable is also applicable to compounds, which canundergo condensation polymerization and form a linear or crosslinkedpolymer.

The term “water soluble” generally refers to solubility of a compound inwater wherein the compound has a solubility of greater than 5 g/100 g,preferably greater than 1 g/100 g in water or buffered water solutions.

The term “water insoluble” generally refers to solubility of a compoundin water wherein the compound has a solubility of less than 5 g/100 g,preferably less than 1 g/100 g in water or buffered water solutions.

The term “imaging agent(s)” or “visualization agent(s)” includes anymedical imaging agent that helps to visualize the human body/tissueusing naked human eye or using machine assisted viewing. The termgenerally applies to but not limited to: coloring compositions thatinduce color to medical devices and drug delivery compositions (asdefined above), radio-opaque contrast agents that help to visualizeorgans/tissues using x-ray imaging techniques, NMR contrast agents thatassist in MRI imaging techniques and the like.

The term “cavity” is defined as an empty space or void in an otherwisein the live tissue or bioprosthesis tissue. The cavity may be filledwith injectable compositions, biological fluids, air or gas. Also, the“cavity” that is within a medium, such as live or prosthetic tissue, orother medium may be a “formed cavity” that is formed into the medium sothat the “cavity” remains in the medium after formation. The medium mayalso be a gel, hydrogel, or other medium that can retain a “formedcavity” by the processes described herein. Once the “cavity” is formed,the medium becomes a “cavity-containing medium.”

The term “porosity” is defined as the presence of pores, voids,cavities, grooves, pockets and indentations within a tissue. The phrases“creation of artificial cavities” and “creation of artificialporosities” have been used synonymously in this application and mean thesame.

The term “cell/s” are defined as mammalian cells that can be grown asprimary cultures as well as established mammalian cell lines, includingtransformed cells. Stem cells which can be converted into any type cellswhen provided with proper biological or chemical stimulus are mostpreferred. The cells include but are not limited to human foreskinfibroblasts, pancreatic islet cells, dopamine secreting ventralmesencephalon cells, adrenal medulla cells, beta cell insula's,lymphoblastic leukemia cells, T-cells, Chinese hamster ovary cells,mouse 3T3, fibroblasts and neuroballistic cells and the like. Mammaliancells obtained from various organs such as brain, kidney, heart, liver,skin, pancreas, intestine, lung, muscle, artery, immune cells and thelike. Additionally, therapeutic enzyme systems, therapeutic bacteria,therapeutic virus, therapeutic genes, hormones, and retroviruses forgene therapy may be referred as cells.

The term “unibody” is defined as a solid mass which when pushed at oneend from the device, is pushed out at the other end without breaking orsubstantially changing its shape. An example of unibody is solid PLGA orHDPE plastic cylinder when pushed out from one end of the device, comesout at the other end as a cylinder. A loose dry powder filled inside thedevice is not considered as unibody implant as some of the powderparticle may stay in the device. However, the same particles may beencased or encapsulated in a hydrogel or other material and can thenform a unibody implant which may be pushed out from the device into thebody as a unibody implant. The same particles may be partially orcompletely fused or sintered to form a unibody. The same particles maybe bound using an adhesive or other binders to act as a unibody implant.The term “biodegradable unibody forming matrix” is defined as anybiodegradable compound including biodegradable polymers and non-polymerssuch as sugars that has capability to from a unibody microimplant. Insome instances a “unibody” may contain cracks, fissures, separations, orimperfections, but when formed as described herein may be considered tobe a “unibiody.”

The present invention is now described with reference to the drawings.

FIGS. 1A, 1B and 1C show partial and schematic representation of in situgenerated drug delivery implant made using conventional syringe basedmethod and array based methods described in this invention. FIG. 1Ashows an injectable composition that is injected as a crosslinkableprecursor fluid/liquid from a conventional syringe using intramuscularinjection into muscular tissue (1003). The precursor liquid forms a gelor polymer in situ inside the intramuscular tissue as a single solidimplant having an irregular shape. The formed implant may have drug orcells entrapped in the implant. As depicted in FIGS. 1A, 1B, the implant(1004 or 1005) is created without creating any artificial cavity priorto injection. FIG. 1B shows an injectable composition comprisingbiodegradable microspheres with drugs (1005) and FIG. 1A showsencapsulated microspheres with cells (1004) injected into musculartissue (1003). Some of the microspheres/cells in the 1004 or 1005implants, typically the middle portions of 1004 or 1005, are in contactwith itself and not with the surrounding tissue. This can potentiallyaffect the in vivo drug release profile. This isolation of the implantfrom the muscular tissue can prohibit cells to get required nutrientsfrom the tissue thereby potentially reducing cell viability.Microimplant array with drugs or cells (1006) formed using methods,compositions and apparatus described in this invention is shown in FIG.1C. The microimplant array shows well defined shape and severalmicroimplants are formed, hence providing large surface area. Due touniform separation of microimplants, each microimplant is surrounded bya tissue enabling better drug diffusion and also helps access nutrientsfor the cells from the tissue.

A partial and schematic representation of making in situ generated drugdelivery array for sustained drug delivery is shown in FIG. 2. A partialschematic of skin tissue is represented by epidermis (1001) and dermis(1002) layers. Artificial porosity is generated in the epidermis and/ordermis layer by many methods known in the art or described in thisinvention. Artificially created cavities in the skin tissue areschematically shown as conical shaped cavities (2001), as anillustrative example. The cavities (2001) are then filled with fluidinjectable drug delivery composition/s comprising drug/s or bioactivecompound/s or live cells (2002). Optionally the fluid composition isconverted into solid or semisolid or hydrogel (2003) by physical and/orchemical means and entrapping the drug/cells in the in situ formed solidor gel. The drug is released from the solid or gel in the surroundingtissue by diffusion and/or biodegradation or combinations thereofprocesses. Live cells in the array can also perform therapeuticfunction.

A partial and schematic representation of making in situ generated drugdelivery array for sustained drug delivery is shown in FIG. 3. Skintissue (1001) is first covered with a fluid drug delivery compositionsuch as 10 percent PLGA and coumarin solution (3002) in DMSO (coumarinis added as model drug, ten percent relative to PLGA plus drug weight).A metal, polymer or ceramic microarray comprising of needles with sharpedges (3001) and backing material (3005) is placed on the skin tissuecovered with the polymer solution (3002) and is pressed against theepidermis (1001) and dermis (1002) layers to perforate the skin. Duringthe perforation step, the needles of the microarray create artificialcavities and also carry the drug delivery composition in the cavities(3003). The microarray needles may be withdrawn or are dissolved away inthe skin/body creating an artificial porosity which is then filled bythe drug delivery composition. Optionally the fluid composition isconverted into solid or semisolid or hydrogel (3004) by physical and/orchemical means and entrapping the drug in the in situ formed solid/gelmatrix. The drug is released from the solid 3004 in the surroundingtissue by diffusion and/or biodegradation or combinations thereofprocesses.

A partial and schematic representation of making in situ generated drugdelivery array for sustained drug delivery is shown in FIG. 4. A partialschematic of skin tissue is represented by epidermis (1001) and dermis(1002) layers. Artificial porosity is generated in the epidermis and/ordermis layer by using dissolvable microneedle array (4001). The array(4001) is made using hyaluronic acid or dextran and the like. Thedissolvable array (4001) is pushed in the tissue and needle materialsare allowed to dissolve in the body or tissue. The cavities created bythe dissolution of needles (4002) are then filled with fluid injectabledrug delivery composition/s comprising drug/s or bioactive compound/s orlive cells (4003). Optionally the fluid composition is converted intosolid or semisolid or hydrogel (4004) by physical and/or chemical meansand entrapping the drug/cells in the in situ formed solid or gel. Thedrug is released from the solid or gel in the surrounding tissue bydiffusion and/or biodegradation or combinations thereof processes.

FIG. 5 shows a partial schematic representation of a method of creationof cavities using microarray of coated hollow needles and filling thecavities with an injectable composition. The coating or pluggingprevents tissue coring during the use of hollow microneedle based array.5001 depicts a hollow microneedle of an array such as 33 MP array. Thetip of needle (5001) is coated with water dissolvable coating/plug orremovable coating (5002). The needle tip also can be plugged with awater dissolvable or removable plug (5003). The coated needle isinserted in the skin tissue (1001 and 1002). The coating or the plugprevents insertion of tissue and other material in the inserted area ofthe needle and maintains the hollow space (5004) or cavity inside theneedle. The needle is inserted in the tissue and is then filled with aninjectable composition such as fibrin sealant, DuraSeal sealant orbiodegradable polymer solution in water miscible biocompatible solvent(5005) with drugs and/or cells. The water in the tissue or components inthe injectable material dissolve the coating 5002 or plug 5003 whichenables removal of the needle from the tissue without obstruction fromthe coating/plug material. The injectable composition may undergophysical or chemical changes forming solid implant 5006 in the tissue.The needle may be removed after the solid implant is formed.

FIGS. 6A and 6B show partial schematic representation of “array inarray” apparatus useful for forming microimplant array in the skin ortissue. FIG. 6A comprises of Panels 6A-1, 6A-2, 6A-3-1 and 6A-3-2. FIG.6B comprises of Panels 6B-1, 6B-2, 6B-3-1 and 6B-3-2.

The apparatus has two parts namely a bottom “base array” and a top“plunger array”, both schematically shown in FIG. 6A and FIG. 6Brespectively. The base array has a base plate with a sharp hollowmicroneedle array protruding perpendicularly from one of the surfaces ofthe base plate. The base array may also contain the microneedle array,and may be referred to as the microneedle array. The top plunger arrayalso has a base plate, designated as plunger plate, with solid needles(e.g., sharp or unsharp, which may be shafts, plungers, plunger shafts,or other) protruding perpendicular to the plunger plate. Thearrangement, length/size and shape of the plunger and base array needlesis identical except that the plunger array needle fits smoothly insidethe hollow cavity of the base array needle and can move freely insidethe cavity up and down. This is achieved since the diameter of theplunger array needles is lesser than the diameter of the base arrayneedles. Panel 6C-1 shows the plunger array on top of the base array,with centers of both corresponding needles coaxially aligned such thatthe plunger array is disposed within but not completely inserted in thebase array. The spacer lock prevents the plunger array from beinginserted completely. Panel 6C-2 shows the plunger array on top of thebase array inserted via guiding posts completely after removal of thespacer lock. Plunger array needles occupy space in the base arraycavity. Panel 6D-1 shows base array cavities filled with preformed or insitu generated microimplants with drug and/or cells and is ready forimplantation. Panel 6D-2 shows insertion of both the arrays in the skintissue and the base array cavities are occupied by plunger array needlesand the implants in the base array cavities are pushed into skin tissue.Both the arrays are subsequently removed leaving behind the implantarray with drug/cells in the skin tissue. Panel 6A-1 shows a schematictop view of the base array with hollow microneedles and optional fourguide posts for ease of insertion and alignment of needles of base andplunger arrays respectively. The base array base plate has length l,width w and thickness t. It also shows number of needles (n) in an arrayformat (5 by 5 hollow microneedles, 25 total needles, n equals to 25) inthe base array base plate with average needle cavity diameter d. Theproximal end of the hollow needle has opening on plate surface withaverage needle internal diameter d. The average needle diameter at thedistal end is d1. The five needle rows are identified as R1, R2, R3, R4and R5 and five columns are identified as C1, C2, C3, C4 and C5. Eachneedle in the array is identified by the respective column and rownumber. The first needle is identified as R1C1 and middle needle isidentified R3C3 and other needles are identified in a similar manner.The distance between each needle is denoted by a and hollow needlesprotruding from the base plate surface with fixed length is shown as b.Optionally the base plate has four guiding posts with diameter c andheight h which enables smooth insertion of the plunger array in the basearray. The guiding post also helps to hold/grab the base array duringits use and tissue insertion. Panel 6A-2 shows the base array with sideview wherein hollow needles protrude from the base plate at 90 degreeangle. Panel 6A-2 shows base plate having thickness t and hollow needlelength b and external average diameter of needle e and internal averagediameter is d. Panel 6A-3-1 shows an expanded view of one of the hollowneedles where the needle has a sharp edge and cut at (α) degree angle(30 degree in this illustrative case) for ease of insertion in thetissue and average internal cavity diameter at distal end is d1. Thecavity volume/space in the needle is shown as β. The Panel 6A-3-2depicts an alternate embodiment wherein the hollow needle volume/space βis partially or completely occupied by a microimplant 6013 (6013comprises drug and/or live cells and the shape of the implant iscylindrical or conical with sharp needle edge). Preferably 6013 is aunibody implant.

Panel 6B-1 shows a schematic top view of the plunger array with solidmicroneedles and optional four guide holes for ease of insertion andalignment of needles from base and plunger array. The holes in theplunger array and guide posts on the base array are at the samecorresponding location on respective base plates. The center of guideposts on the base array matches with the center of guide holes onplunger array. The plunger array has a plunger plate with length l′,width w′ and thickness t′. It also shows an exemplary 25 number ofneedles (n′) in an array format (5 by 5 hollow microneedles in arrayformat, 25 total needles, n′ equals to 25). Each needle in the array isidentified by its row and column number. The distance between eachneedle is a′ and length of the needle protruding from the plunger plateis b′. Optionally the base plate has guiding holes with diameter c′which are slightly larger than guiding post diameter of base array (c)which enables smooth insertion of the plunger array in the base array.Panel 6B-2 shows plunger array in Panel 6B-1 with side view whereinsolid plunger array needles are protruding from the base plate at 90degree angle.

Panel 6B-2 shows the plunger plate having thickness t′ and needle lengthb′ and external average diameter of the needle e′. Panel 6B-3-1 showsexpanded view of one of the solid plunger array needles where needle hassmooth cylindrical non-cutting shape designed for pushing the 6013implant with length b′ and diameter e′. The Panel 6B-3-2 depicts analternate embodiment wherein the plunger array needle has passage/hollowtube (6022) in the needle and base plate for transfer of injectablecomposition in the base array needle cavity. The injectable compositionis transferred via an injection port (6023) attached to base plate ofplunger array via passage 6022 into base array needle cavity. Ifdesired, a syringe with injectable composition may be connected via port6023 to fill the base array cavity via 6022 passage. The composition ispushed from the syringe in the cavity. Panel 6C-1 shows schematic sideview of plunger array positioned on top of base array, but not inserted.6003 denotes the base plate of the base array. 6016 denotes the plungerplate of the plunger array. 6019 denotes the solid microneedlesprotruding from the plunger plate. 6007 denotes the hollow microneedlesprotruding from the base plate. 6008 denotes the guiding posts. Theguidepost bars of base array are inside the holes of plunger array. Thisensures alignment of center of base array needles with center of plungerarray needles. This alignment is important to insert all plunger arrayneedles entering in base array needles at the same time. The insertionof plunger array needles in the base array hollow cavity needle isprevented by a spacer lock 6024. As shown in Panel 6C-2, spacer lock isremoved and the plunger array needles are pushed inside the hollowcavities of base array. The plunger array plate is on top of base arrayplate. Panel 6D-1 shows schematic side view of base array similar toPanel 6A-2 except the hollow cavities are occupied by prefabricated orinsitu generated microimplants (6013) with cells/drugs in the arraycavities. Panel 6D-2 shows schematic side view of base array and plungerarray wherein the plunger array has pushed the implant from (Panel 6D-1,6013) out of base array cavity into the skin tissue (1001 and 1002). Theimplants 6013 in the form of an array are left in place for therapeuticeffect after withdrawal of both the arrays from the tissue.

A partial and schematic representation of an in situ generated drugdelivery array comprising drug encapsulated microparticles is shown inFIG. 7. A partial schematic of skin tissue is represented by epidermis(1001) and dermis (1002) layers. Artificial porosity is created in theepidermis and/or dermis layer as conical shaped cavities isschematically shown as 7001. The cavities (7001) are filled with fluidinjectable drug delivery compositions comprising microparticlesencapsulated/coated with drugs (7002), preferably the composition ormicroparticles is colored or fluorescent. The drug is released from themicroparticles in the cavities and in the surrounding tissue in asustained manner. FIGS. 8A, 8B, 8C, 8D and 8E show representative imagesof cavities formed in the tissue and gelatin gel and then filled withpolymers with drug and/or visualization agent. A microimplant array isformed in the model tissue like material (gelatin gel, 8001) and sheepskin tissue (8005) or pericardial tissue (8008). A 3 by 3 array (33 MP)is used to create porosity in transparent gelatin gel (8001) which isthen filled with PLGA polymer containing methylene blue as a colorantand/or drug. The precipitated PLGA polymer and its blue color in 3 by 3microimplant array form (8002) is shown in FIG. 8A. FIG. 8B showsgelatin gel with 3×3 microimplant array made from PLGA polymer solutionand coumarin as fluorescent dye using 33 MP array. The PLGA implantarray formed in situ which is fluorescent under blue light (8003) isshown in FIG. 8B. A PLGA polymer with coumarin microimplant array wereformed by direct injection in the sheep dermal tissue (8005) using 33 MParray at 3 separate locations is shown in FIG. 8C. The formedmicroimplants arrays are fluorescent under blue light (8004). FIG. 8Dshows microcavities (8009) created in pericardial tissue (8008) beforeinfusion of injectable composition. FIG. 8E shows sheep skin tissue(8005) infused with 4×4 array (8007). The array 8007 is made by infusingPLGA polymer solution containing magnesium carbonate stained with eosin.The array 8007 is pictured under blue light wherein eosin in themicroimplant array formed is fluorescent.

FIG. 9A shows a representative image of an iron containing implant 9001formed in situ inside the tissue using methods described in thisinvention. FIG. 9B1 depicts the image of 10×10 array (9002) made fromhyaluronic acid salt and iron pyrophosphate formed by casting insilicone rubber mold and FIG. 9B2 shows microscope image of one of theneedles of array (9003) shown in FIG. 9B1. The FIG. 9B2 shows sharpneedle tip of one of the needles of array 9003.

FIG. 10 shows cumulative moxifloxacin base release profile from the 2 cmby 2 cm sheep tissue prepared according to Example 14B describedsubsequently in this application. The porosity was first created using ametal microneedle array in the tissue and the cavities created were thenfilled with biodegradable polymer (PLGA) solution in NMP comprisingmethylene blue as colorant and moxifloxacin base as a drug.

FIG. 11 shows cumulative moxifloxacin base release profile from the 2 cmby 2 cm sheep tissue prepared according to Example 14C describedsubsequently in this application. The implant array was prepared byapplying a metal microneedle array through a layer of biodegradablepolymer (PLGA) solution in NMP comprising methylene blue as colorant andmoxifloxacin base as drug. FIG. 12 shows cumulative moxifloxacin baserelease profile from the 2 cm by 2 cm sheep tissue prepared according toExample 14A described subsequently in this application. The implantarray was prepared by direct injection of biodegradable polymer (PLGA)solution in NMP comprising methylene blue as colorant and moxifloxacinas drug. The injection was made using hollow microneedle array device(33 MP).

FIG. 13 shows a drug release profile of bupivacaine base frommicroimplants array formed in the tissue using oscillating needle. Theartificial cavities are formed through the polymer solution on thetissue surface by the use of oscillating needle and not a microneedlearray. The polymer solution was driven by the oscillating needle in thebovine pericardium precipitates in the cavities which encapsulates thedrug and releases it in a sustained manner. The lower curve (solidsquares) is for the control sample which is infused without drug. Asexpected the bupivacaine was released in a sustained manner from the insitu formed implant. The control sample did not show bupivacainerelease, as expected.

FIG. 4 shows a drug release profile of rifampin encapsulatedmicrospheres from microimplants array formed in the tissue using amicroneedle array. The artificial cavities are formed through therifampin microspheres suspension in glycerol on the sheep skin tissuesurface by the microneedle array. The array needles are pressed on thetissue through the suspension. As the needle penetrates the tissue andform a cavity, the suspension is carried along with it. The glycerol isdissipated in the tissue leaving behind microspheres in the artificialcavities. Microspheres without rifampin were also incorporated in thetissue and used as a control. Rifampin release profile from the tissueand from the control lower curve (solid circles) is shown. As expectedthe rifampin microspheres showed a sustained release of rifampin in thetissue and control microspheres did not show rifampin release (solidcircles, bottom curve). The trace amount of drug in one data point incontrol sample is believed to be due to contamination or of unknownorigin.

FIG. 15 shows an illustrative “array in array” apparatus as described inFIGS. 6A and 6B. FIG. 15A shows a base array 1501 with top view showing5 by 5 hollow microneedle array created in stainless steel metal plate.Base array plate length and breadth is 20 mm and thickness is 1 mm.Outside diameter (OD) of the hollow microneedles 1502 is 0.55 mm whileinternal diameter (ID) of cylindrical cavity is 0.31 mm. The proximalend of the needle has opening on the base plate with ID 0.31 (same asneedle ID, d) and the other end of the needle (distal end) has a sharpedge and ID of 0.31 (d1=0.31). The base array plate has 4 guiding posts1503 with diameter 2.48 mm. Distance between each needle is 2 mm. FIG.15B shows the side view of same base array 1501 showing base metalthickness and hollow sharp microneedles (at distal end) protruding outof the base plate 1504 surface. Total length of hollow needle is 2 mm ofwhich 1 mm is inside the base plate and 1 mm is protruding out of baseplate. The outer needle edge is cut at 30 degree angle for ease ofinsertion. FIG. 15C shows plunger array 1505 with top view showing 5 by5 microneedle array with solid plunger needles 1508 (also may bereferred to as shafts, plungers, plunger shaft, which may be blunttipped, but may also be sharp) created in stainless steel metal. Lengthand breadth is 20 mm and thickness is 3 mm. Outside diameter (OD) of thesolid microneedles is 0.3 mm which is smaller than the base array cavityID (0.31 mm). The plunger array plate 1506 has 4 guiding holes 1507 w z1000ith diameter 2.51 mm which is slightly larger than guiding postsdiameter (2.48). Distance between each needle is 2 mm. FIG. 15D showsthe side view of the plunger array 1505 showing base metal thickness andsolid needles protruding out of the bottom surface of the plunger arrayplate 1506. FIG. 15E shows the plunger array 1505 placed on top of thebase array 1501 (not inserted but aligned and ready for insertion)wherein each center of each needle of top array is aligned with centerof base array needle. The holes of plunger array are aligned withguiding posts of the base array. FIG. 15F depicts the position whenplunger array needles are completely inserted in cavities of base arrayneedles.

FIG. 15G shows PLGA based cylindrical implant with coumarin as modeldrug and fluorescent agent is formed in situ inside hollow cavities ofbase array first and then pushed inside gelatin gel using plunger arrayas shown in FIGS. 15E and 15F. The green fluorescence of PLGA polymermicroimplant array (5×5 array) formed is clearly visible under bluelight due to coumarin fluorescence. FIG. 15H shows catgut suture basedcylindrical microimplants with fluorescent coating is created first. Thepreformed implants are then placed in hollow cavities of base array andthen inserted in the sheep skin tissue using plunger array as describedabove. The inserted microimplants show green fluorescent coating on theouter edge of the implant under blue light. The apparatus used in makingarrays (FIGS. 15G and 15H) is one of the several porotypes made and usedto make implanted microarrays.

FIG. 16A shows a photographic image of part of human nail withartificially created four cavities (average diameter around 700microns). FIG. 16B shows FIG. 16A cavities filled with PLGA basedbiodegradable composition with D and C violet as a colorant. FIG. 16Cshows in vitro terbinafine hydrochloride (an exemplary antifungal drugsuitable for treatment of fungal nail infection) release profile fromPLGA based experimental composition from the implanted microarray.

FIG. 17 shows bupivacaine base release profile of coated threads.Submucosa twisted threads were coated with PLGA polymer and bupivacainebase. Bupivacaine base release from the control (no drug, polymer only,triangles), 20 percent coating (solid circles) and 50 percent coating(rectangles) is shown in FIG. 17. As expected, the control sample didnot show any significant release of bupivacaine. The threads coated with20 and 50 percent drug solution provides sustained release ofbupivacaine up to 72 hours. The coated fibers or cylindrical threads canbe cut/sectioned to form bupivacaine based coated microcylinders ofsuitable length, which can be used as preformed microimplants to make animplanted microarray using AIA device as described in this invention.

FIG. 18 shows a partial schematic representation of microneedle arraycomprising the islets of Langerhans implanted in the skin tissue. 1801shows an array of conical needle shaped artificial cavities in the skintissue which are filled with insulin producing cells (islets ofLangerhans) encapsulated in a semipermeable biodegradable or biostablepolymer/hydrogel matrix (1802). The cells can exchange through thesemipermeable matrix insulin, glucose, nutrients from the surroundingskin tissue for survival and metabolic waste products. The live cellscan survive and produce insulin based on glucose concentration presentin the skin tissue fluids. 1803 shows microencapsulated islets cellmicrospheres filled in the cavity 1801. 1803 are islet cells inside themicrosphere and 1804 is spherical shaped semipermeablemicroencapsulation microsphere matrix which enables exchange ofnutrients and cellular waste products but prevents immunoglobulinsdiffusion and offers immunoprotection.

FIGS. 19A and 19B show exemplary images of microimplant arrays createdusing inventive methods and illustrative devices used to create sucharrays. FIG. 19A shows image of 4 by 4 microimplant array made in thesheep skin. Array is an exemplary synthetic biodegradable crosslinkedhydrogel gel (1901, white colored) containing magnesium carbonateencapsulated microparticles as visualization agent or as a biodegradablefiller or as an exemplary drug encapsulated microparticles. FIG. 19Bshows image of 10 by 10 liquid microimplant array made in sheep skin.Array is an exemplary liquid carrier vitamin E acetate containing teastained magnesium carbonate (1902, red colored) added as visualizationagent. The array is liquid at ambient/body temperature.

FIG. 20 shows iron release profile of samples prepared according toExample 8. The ferric pyrophosphate particles are suspended PLGAsolution in NMP and tattooed into sheep dermal tissue. Control sample istattooed without ferric pyrophosphate. The release of iron from ferricpyrophosphate treated samples (rectangles) and control samples (noferric pyrophosphate, solid circles) is shown in FIG. 20.

FIG. 21 shows a method for in situ implant formation in the human oranimal body comprising biodegradable fillers. 2101 schematicallyrepresents an injectable composition comprising a drug and biodegradablepolymer in water miscible organic solvent or crosslinkable precursorcomposition/s comprising a drug or a thermoreversible polymercomposition in aqueous solution or polymer melt. 2102 comprisesbiodegradable, biocompatible inorganic or organic filler microparticlesthat are insoluble in the injectable composition 2101. The components of2101 and 2102 are mixed to form a suspension/emulsion 2103 and injectedinto human or animal body via conventional syringe or using methodsdescribed in this invention to form implantable arrays. The injectedcomposition undergoes physical and/or chemical change (precipitation,crosslinking, cooling, thermoreversible gel formation and the like)entrapping the drug and the filler in the formed gel/solid implant. Thepresence of filler is believed to provide nucleating sites for polymerprecipitation as well as provide more surface area for the implantthereby altering drug release profile. Filler also changes themechanical properties of the in situ precipitated polymer/gel whichhelps to push out from “array in array” apparatus described in thisinvention. FIG. 21A shows the steps involved in making the implant withfiller and FIG. 21B shows release profile of bupivacaine hydrochloridefrom the in situ made PLGA array implant with and without magnesiumcarbonate as an exemplary filler.

FIGS. 22A, 22B, 22C and 22D show exemplary schematic/images ofmicroimplant arrays created using inventive methods and illustrativedevices according to the present invention. FIG. 22A shows microneedlearray (2201) containing 20 microneedles (2202) used to create 20 microcavities per insertion in the tissue. FIG. 22B shows 33 MP hollowmicroneedle array with 3 by 3 hollow microneedles array (2204) attachedto a syringe via Luer hub (2203) containing injectable composition(2205, PDLG 5002 biodegradable polymer solution in DMSO with methyleneblue as a visualization agent) to form microimplant array. FIG. 22Cshows a 3 by 3 array of fluorescent biodegradable cylindrical rods(2206, 100 microns diameter and 1000 microns height prepared by slicing100 microns diameter fluorescent fiber/thread) and is inserted in thetissue to form an array.

FIG. 22D shows image of 4 by 4 microimplant array made in sheep skin.Array is an exemplary synthetic PEG-polylactone based biodegradablethermosensitive polymer hydrogel containing rifampin encapsulatedmicrospheres (2207, red colored) for sustained drug delivery.

FIG. 23A to C show schematic representation of use of expandable arrayneedle in forming drug delivery microimplant array. 2301 is anexpandable needle/stent with hollow cavity for storage of drug/celldelivery microimplant and may have sharp edge at distal end. Plungerarray needles used in AIA device described in this invention can beexpandable needles such as 2301. The needle is present in the compactform in the base array cavity needle of AIA device. The base arraycavity space prevents the expandable needle from expansion. FIG. 23Ashows an expandable needle in compact form with microimplant (6013) inits cavity and is pushed out from the base array cavity into the skintissue in unexpanded form but with implant in its cavity. FIG. 23B showsthe expansion of needle/stent into an expanded shape or its memorizedshape (2302). The expanded shape has been pre-memorized intoneedle/stent using a heat treatment of the Nitinol alloy. The expandedshape release the implant in the skin tissue and needle 2301 is thenwithdrawn in the base cavity array in compact form and then out of theskin tissue (FIG. 23C). Preferably during expansion of needle, theimplant is pushed out in the skin tissue. The microimplant (6013) isleft in the tissue in an array format for therapeutic action.

FIG. 24 shows a partial schematic representation of another version of“array in array” device wherein a separate cartridge for holdingmicroimplants is used. The cartridge can be aligned and placed betweenbase array or outer array and plunger or inner array. The microimplantsin the cartridge are then inserted into skin/tissue via base arraycavities. FIG. 24A shows an exemplarily circular shaped cartridgewherein cartridge has a base plate (2401) with one or moreholes/cavities (2402). The holes have openings on both sides of theplate 2401 surface (proximal and distal end). The holes 2402 can befilled with preformed or in situ formed microimplants (6013) withdrug/cells (A2). The bottom and/or top surface of 2401 may be coveredwith protective cover (2407) which may be removed at the time of use.The 2407 prevents unwanted slippage of implant from the holes duringstorage and handling. B1 and B2 represent base array and plunger arrayrespectively similar to described in FIG. 6 wherein 2405 is a base plateto which hollow sharp needles 2404 are attached. Proximal end of needleshas opening on the base plate to load microimplants. B2 is similar tothe plunger array described in FIG. 6 wherein 2405 is a base plate towhich solid non-cutting needles (2406) are attached. The internaldiameter of hollow needles (2404) is same as hole diameter in cartridge(2402). The external diameter of plunger needles (2406) is less than thediameter of holes (2402) and it can freely move up and down in theholes/cavities of 2402 and 2404. The number of needles and holes andtheir arrangement in the array is identical in A1, B1 and B2. FIG. 24Cshows array B1 inserted in the skin tissue and cartridge A2 withmicroimplant is placed on top of array B1 with protective cover 2407removed. The center of all the holes in FIG. 24A is aligned with thecenter of hollow needle opening on baseplate of B1. The center ofplunger array needles in FIG. 24C is also aligned with center of holesin A2 and B2 but is not inserted in cartridge A2. FIG. 24D shows theinsertion of plunger needles in the holes of cartridge A and cavities ofB1 and pushing the implants from A2 via cavities of 2404 in the skintissue. Both the arrays and cartridge is pulled from skin tissue leavingbehind microimplant array with drug/cells for local or systemictherapeutic effect. The cartridge may be packaged and stored separatelyand used as described above or it may be packaged in the pre-alignedform in the AIA device and used for implantation. The cartridge, plungerarray may have additional holes (not shown) and base array may haveguiding posts (not shown) to help in alignment similar to described inFIG. 6.

FIG. 25 shows partial schematic representation method to make base orplunger array as described in FIG. 6. Metal/plastic/ceramic preferablymetal hollow tubes with a desired diameter and length are provided(2501). The tubes are encased in a plastic or metal plate (2502) via insitu casting of plastic resin or injection molding or wielding/adhesivebonding or other methods. The encasing of tubes acts as a circular baseor plunger plate described in FIG. 6. The encased tubes are cut on thebase plate surface (proximal end, straight cut) and angular cut atdesired angle at distal end to produce sharp edged (2503) hollowmicroneedles at distal end. The sharp-edged microneedles (2503) protrudefrom the base plate (2502). The cut edges may be polished to produce asharp edge. The opening on base plate surface (proximal end, not shown)is used for insertion of microimplant for forming/casting in situimplant. The hollow tubes may be substituted with solid rods to produceplunger array. Alternatively, plunger array can be entirely made byinjection molding of commonly used medical thermoplastics.Alternatively, the needles may be first cut to desired length with sharpend and then encased in a plastic/metal/ceramic base plate (2502) toproduce needles with sharp edges at distal end and opening in proximalend.

FIG. 26 shows partial schematic representation of various configurationsof biodegradable metal based, preferably magnesium alloy based,microneedles and arrays that can be useful in making implantable drugdelivery devices. FIG. 26A schematically shows a biodegradable magnesiumalloy based hollow array needle (2601) with sharp distal edge (2602) foreasy tissue penetration. The hollow cavity of needle is partially orcompletely filled with sustained drug delivery composition such as PLGApolymer with a drug like rifampin (2603). FIG. 26B shows a schematic ofan illustrative array needle 2604 whose external surface is coated withbiodegradable sustained drug delivery composition (2605). Thecomposition 2605 may have one or more coating layers and one of them maybe a release rate controlling layer without a drug. FIG. 26C shows ahybrid needle wherein the tip of the needle (2606) is made up usingbiodegradable metal for easy skin penetration and the drug deliveryportion (2607) is made biodegradable polymer/hydrogel with sustaineddrug or live cell delivery composition. FIG. 26D shows schematics ofillustrative biodegradable metal array needle with variousconfigurations for infusing drug delivery compositions. FIG. D1 shows abiodegradable metal array needle (2608) wherein wedge shaped micropockets are created inside the needle surface and then filled with drugdelivery composition (D1, 2609). In another variation (D2), rectangularportions have been cut out in the needle body to create a space for drugdelivery composition filling material (2610). In another variation (D3),several artificial microcavities of various shapes (cylindrical in thisillustrative case) or holes are created in the needle surface/body andthe cavities/holes are then filled with injectable drug deliverycompositions (2611). FIG. 26E shows illustrative microneedle implantablearray device with biodegradable metal based microneedles attached to aflexible removable backing material. Four microneedles havingholes/cavities filled with biodegradable drug delivery compositions(2611, D3) are attached using a pressure sensitive adhesive (2613) toflexible backing material (2614) in an implantable array format tocreate a biodegradable microneedle array based drug delivery device. Theneedle base is attached to the adhesive and free distal end with sharpedge is used for tissue penetration. The device is inserted in the skintissue by pressing the backing layer with needles sharp ends facing theskin and implanted in the skin. The backing/adhesive material is removedleaving behind the array in the skin. The metal array and drug deliverycompositions are biodegradable and provide a drug for local or systemictherapeutic effect.

This invention teaches methods and compositions for infusing injectablecompositions, preferably with drug/cells or imaging agents in the bodyor skin or in the tissue of a bioprosthesis.

Different embodiments of the present invention are described byreferring to various medical/industrial applications and examples asprovided.

Description of Preferred Embodiments

In this invention, the microimplant array, preferably biodegradablearrays are made in situ inside the tissue rather than in a controlledfactory setting. Briefly, artificial porosity or microchannels orcavities are first created inside a live tissue or bioprosthesis tissueusing a surgical procedure such as laser drilling or oscillating needleor microneedle array and the like or any other method known in the artor yet to be discovered. The pores or microchannels or cavities createdby the microarray are then filled with an injectable drug deliverycomposition comprising a drug or bioactive compound or live cells. Inpreferred compositions, the drug and biodegradable carrier matrix areinjected in a fluid state and cast or solidified in situ inside thepores to form microarray or microimplant like structure inside thetissue. Preferably the solid formed is a unibody implant. The solidifiedcompositions in array form release the drug in a sustained manner forlocal or systemic therapeutic effect. In some embodiments, usingspecialized devices described in this invention, microimplant arrays aremade in situ in the tissue using prefabricated microimplants. Thecomparison of conventional microarray based drug delivery systems andmicroarray like structures created inside the skin or tissue asdescribed in this invention is shown in Table 1.

TABLE 1 Comparison of drug delivery arrays made externally and in situgenerated array as described in this invention. Conventional Drug InSitu Generated Drug Delivery Array Delivery Array Externally fabricated.In situ generated. Limited in shape and No need to have sharp materialchoice. edges. The array shape Generally, must have is dependent onskin/ sharp edges for easy tissue and artificial penetration in the skinporosity/cavity shape tissue. created for the specific application.Array material must be Can form liquid based a solid for implantationmicroimplant arrays. and cannot be liquid in nature. Generally, hasexternal Not applicable. backing material for application of externalforce and ease of insertion. Cost associated with Cost associated withfabrication of array in porosity generation in factory setting and thetissue and preparation insertion mechanism. and injection of injectablecomposition. The microneedle array No such requirement which materialmust have hardness enables to choose from wide and mechanical strengthvariety of biocompatible to withstand resistance materials. fromskin/tissue for insertion. Soft elastomeric materials are notpreferred/suitable. Limited availability of More flexibility on size anddose due to microimplant size and drug external manufacturing. dose. Thesize, number of microimplants in the array and their arrangement can betailored for specific clinical application.

Biodegradable implants can be made in situ for drug deliveryapplications (U.S. Pat. No. 5,567,435 cited herein for reference only).Generally, such methods are useful to fill a body cavity that isnaturally present in the body. Though such methods are useful, thesemethods have limitations. A comparison of in situ formation of materialsgenerally known in the art and inventive methods and compositionsproposed in this invention are shown in FIG. 1 and Table 2. FIG. 1 showspartial and schematic representation of making in situ generated drugdelivery implant made using conventional syringe based method and arraybased methods described in this invention. A partial schematic ofmuscular tissue is represented by 1003. The injectable composition isinjected as a crosslinkable precursor fluid/liquid from a conventionalsyringe using intramuscular injection. The precursor liquid forms a gelor polymer in situ inside the intramuscular tissue as a single solidimplant generally with irregular shape (1004) (FIG. 1A). The formedimplant may have drug or cells entrapped in the implant. The implant(1004) is created without creating artificial cavity first. FIG. 1Bshows an injectable composition comprising cell encapsulatedmicrospheres (1004) injected into muscular tissue (1003). Some of themicrospheres/cells in the 1004 or 1005 implants are in contact withitself and not with the surrounding tissue (middle portion of theimplant). The middle portion of the implant is devoid of tissue fluidswhich can potentially affect the in vivo drug release profile or cellviability. This isolation of implant from the tissue can also prohibitcells to get required nutrients from the tissue potentially reducingcell viability. Microimplants drug delivery array (1005) formed usingmethods, compositions and apparatus described in this invention is shownin FIG. C. The microarray implant formed according to this inventionshows well defined shape and several microimplants are formed providinglarge surface area. Due to separation between each microimplant, eachmicroimplant is surrounded by a tissue which enables drug extraction bytissue fluids and also enables to get nutrients for the cells from thetissue.

TABLE 2 Comparison of conventional injectable drug delivery systems andmicroimplant drug delivery array as described in this invention.Conventional injectable In situ generated drug drug delivery systemsdelivery microimplant made using in situ array described in thispolymerization systems. invention Inject generally large Depositsseveral small volume, typically greater volume droplets in well- than 1ml delivered using defined artificial cavities a syringe like device.inside the tissue, typically using a microarray device. No control overshape of Well defined cavity shapes the implant. For example, aregenerally used to form/ intramuscular injection cast an implant. ofinjectable composition generally, forms irregular shape implant.Generally, forms one Generally, forms several large body implant insitu. discrete small volume microimplants. Limited area of the inMicroimplants array made situ formed implant according to this inventionexposed to the tissue for generally have large dispersion of drug in thesurface area due to small tissue. In case of size and large number ofmicrospheres based drug implants. Each delivery systems, eachmicroimplant is in contact injected with tissue which enablesmicrosphere may not be in better diffusion of drug contact with tissueand in the tissue. therefore may have difficulty in drug elution intissue fluids. Can be used with commonly May need a specialized useddevice like syringe device and special and needle. manufacturingprocess. No need to create Must create artificial artificial cavity.cavity generally with well- defined shape and size. Removal of implantis Removal, destruction, generally difficult due laser inducedvaporization to deep intramuscular or drug deactivation is injection andabsence possible if implanted under of visualization agent the skinand/or visualization to locate the injected product. agent can assistremoval.

In this invention, new methods and compositions to create or fabricatedrug delivery devices/arrays in situ inside the live tissue orbioprosthesis tissue are disclosed. Briefly, porosity is firstartificially created on live tissue surface such as skin tissue. Thecavities created in the tissue are then filled with drug deliverycompositions for sustained drug delivery and therapeutic effect. Apartial and schematic representation of making in situ generated drugdelivery array for sustained drug delivery is shown in FIG. 2. A partialschematic of skin tissue is represented by epidermis (1001) and dermis(1002) layers. Artificial porosity is first generated in the epidermisand/or dermis layer by many methods known in the art or described inthis invention. Conical shaped cavities (2001) formed in the skin tissueare schematically shown. The cavities (2001) are then filled with fluidinjectable drug delivery composition comprising drug/s or bioactivecompound/s (2002). Optionally the fluid composition is converted intosolid or semisolid or hydrogel (2003) by physical and/or chemical meansand entrapping the drug in the in situ formed solid or gel. The drug isreleased from the solid or gel in the surrounding tissue by diffusionand/or biodegradation and/or bioerosion or combinations thereofprocesses.

Creation of Artificial Porosity in the Tissue:

The term porosity also includes voids, cavities, holes, surface grooves,indentations, channels, roughness and the like. Artificial tissueporosity is created first and is then used to store or fill thetherapeutic agents or drugs or live cells. Many methods can be used tocreate porosity in the tissue. The porosity creation may involvesurgical procedure, preferably MIS surgical procedure. Generally, up to2-5000 microns, preferably up to 5-1000 microns, even more preferably10-600 microns thick human skin layer is preferred because such tissuecan be accessed easily and potentially does not involve any nerveendings, thus enabling a relatively pain free procedure. For greaterthan 600 microns deep tissue access, topical local anesthetics lotionsor gels or injections may be used to reduce pain in creating artificialporosity. One preferred method involves use of physical means ormechanical methods such as use of metal, ceramic or polymer needles ormicroneedle array or coring needles or biopsy needles to create pores ormicrochannels or cavities in the skin tissue. In one exemplaryembodiment, AdminStamp devices, which contain AdminPatch® MicroneedleArrays attached to an applicator, are used. The AdminPatch microneedlearray is available in variety of needle lengths. For example,AdminPatch® 1500 product has thirty-one 1400 micron tall microneedleslocated within 1 sq. cm circular area. The entire device is 20 mm indiameter and is made of medical-grade SS316L stainless steel. This arrayis attached to a stamping tool which enables easy application of arrayon tissue surfaces and creates porosity. Other microneedle arrays,available commercially, have forty three 1100 micron tall microneedles;eighty five 800 micron tall microneedles; one hundred eighty seven 500micron microneedles and seven hundred fifty two 250 um-tall microneedleslocated within 1 sq. cm circular area. In the exemplary embodiment, anAdminStamp 600 Microneedle Array Device, which contains AdminPatch®Array 0600 microneedle array attached to an applicator with sixlow-profile stainless steel screws, is used. This device has one hundredeighty seven 500 micron microneedles located within 1 sq. cm circulararea. A 2 cm by 2 cm sheep skin tissue is used to create porosity.Briefly, the 10 cm by 10 cm sheep skin portion is cut, hydrated for 2minutes in PBS; shaved to remove all hairs and the AdminPatch® Array0600 microneedle array is applied on the tissue. Upon application ofpressure, the needles penetrate the skin surface, creating one hundredeighty seven 500 micron size holes in the tissue surface. To assistvisualization of holes created, Trypan blue staining dye solution or aPLGA polymer solution in n-methyl pyrrolidone (NMP, 10 percentweight/volume polymer in NMP) containing one percent coumarin (relativeto polymer plus drug weight) as fluorescent dye as well as model drug isapplied. The solution is incubated for 1 minute to 10 hours to enablepenetration inside the cavities created. The excess solution is wipedoff from the skin surface. To remove any surface polymer and dye, thesurface was cleaned and wiped off with methanol which is a nonsolventfor the polymer. In the artificial cavities created by the stamp, thewater in skin tissue dissipates the NMP from the solution, forcing thepolymer to precipitate inside the artificial cavities along withfluorescent dye. The precipitated polymer in the cavity takes the shapeof the cavity and form into polymer solid mass with entrapped dye. Theprecipitated polymer solids are observed using microscope under bluelight. The coumarin is fluorescent under blue light which enables to seethe presence of dye and the polymer in the cavity. Other microneedlestamps were used to create cavities/microarrays inside the tissue withdepth ranges (the needle size, shape and length equals to shape anddepth of cavity). If more cavities are needed, the same stamp is pressedat a different location on the tissue. For example, the same stamp isremoved from the surface and reinserted at 1 to 2000 micron apart fromthe first location of insertion. In this way, the number of cavitiescreated is multiplied and several hundred cavities can be created on theskin tissue.

In another variation of this method, a polymer solution is first appliedon the skin surface and allowed to form a liquid layer of 0.5 micron toseveral mm thick. The microneedle stamp is then applied on the skin viathe liquid layer. The microneedles carry the polymer and drug solutioninside the tissue. In this method, the cavity creation and subsequentinsertion of injectable fluid drug delivery composition is done almostat the same time. The needle surface area helps to drag/carry thesolution inside the cavity space created during insertion. This conceptis illustrated in FIG. 3. Partial and schematic representation of makingin situ generated drug delivery array for sustained drug delivery isshown in FIG. 3. The skin tissue is covered with a fluid drug deliverycomposition such as 10 percent PLGA and coumarin solution (3002) in DMSO(coumarin is added as model drug, one percent relative to PLGA plus drugweight). The metal, polymer or ceramic microarray needle (3001) withsharp edges is placed on the skin and polymer solution, and is pressedagainst the skin to perforate the skin. During perforation, the needlesof the array create a plurality of artificial cavities and also carrythe drug delivery composition in the cavities (3003). Optionally thefluid composition is converted into solid or semisolid or hydrogel(3004) by physical and/or chemical means and entrapping the drug in thein situ formed solid matrix. The drug is released from the solid in thesurrounding tissue by diffusion and/or biodegradation or combinationsthereof processes. The use of polymer solution is for example only.Precursor of crosslinking compositions such as fibrin glue prior togelling or crosslinking, neat liquid carrier of drugs and other fluidcompositions may be forced into cavities and converted into solid and/orgels for drug delivery.

In another embodiment, a dissolvable microneedle array is used to createporosity. Various small compounds (with molecular weight below 2000g/mole)/sugars can be used to make dissolvable microneedles ormicroimplants with drug and these include but limited to: xylitol,sucrose, maltose, mannose, cyclodextrin, stachyose, inositol, mallorol,melitose, iso-maltulose, dextran, lactulose, trehalose, turanose,fructose, icodextrin, raffinos, maltodextrin, glucose, lactose,sorbitol, mannitol, melezitose, palatinit, maltulose and the like orcombinations thereof. Partial and schematic representation of making insitu generated drug delivery array using dissolvable array is shown inFIG. 4. A partial and schematic representation of making in situgenerated drug delivery array for sustained drug delivery is shown inFIG. 4. A partial schematic of skin tissue is represented by epidermis(1001) and dermis (1002) layers. Artificial porosity is generated in theepidermis and/or dermis layer by using dissolvable microneedle array(array is made using hyaluronic acid or dextran and the like, 4001). Thedissolvable array is pushed in the tissue and needle materials areallowed to dissolve in the body or tissue. The cavities created by thedissolution of needles (4002) are then filled with fluid injectable drugdelivery composition/s comprising drug/s or bioactive compound/s or livecells (4003). Optionally the fluid composition is converted into solidor semisolid or hydrogel (4004) by physical and/or chemical means andentrapping the drug/cells in the in situ formed solid or gel. The drugis released from the solid or gel in the surrounding tissue by diffusionand/or biodegradation or combinations thereof processes. Briefly,dissolvable microneedles are used to create porosity in the tissue. Thedissolvable microneedles are designed to penetrate the tissue and aregenerally comprised of a therapeutic drug. Upon insertion, the needlesdissolve in the physiological environment such as present in the humanskin tissue (37 degree C., pH 7.4). The dissolution of the dissolvablemicroneedle array creates a space or cavity in the tissue which is thenfilled with the injectable compositions. The injectable compositionsrelease the drug in a sustained manner. The injectable compositions alsomay undergo physical or chemical changes leading to formation of solid,semi-solid or gel like implant in the cavity. Many methods are known inthe art to prepare dissolvable microneedle implants. Some manufacturersalso supply dissolvable microimplant arrays for research use. Ingeneral, aqueous solutions of biocompatible water soluble salts/smallmolecules or macromolecules/polymers are used to make dissolvablemicroarray implants. Biocompatible, non-toxic materials like sugars(various types), cyclodextrin and its derivatives can be used. Watersoluble polymers likes polyvinyl pyrrolidinone, carboxy methylcellulose, polyvinyl alcohol, hyaluronic acid, dextran, chitosan,carboxymethyl cellulose, and the like may be used to make dissolvablemicroneedle array. Generally, arrays are made by casting the aqueoussolutions of desired materials in the mold of desired size and shape. Acasting mold can be made by the use of photolithography process,mechanical cutting and fabrication tools or other methods known in theart. Materials like silicone rubber are preferred because theirelastomeric nature and chemical inertness. Mold materials like silicon(suitable for photolithographic process), polymethyl methacrylate, nylonand the like may also be used. The mold consists of several cavitiesarranged in the form of an array with desired shapes, volumes and sizessuch as conical shape cavities. The desired array material is poured inthe mold cavity and mold and cavity are generally subjected tocentrifugal force to drive and fill the cavity. The water is generallyevaporated and the array material is removed from the mold and used. Inone illustrative embodiment, a silicone rubber mold is used to prepare10 by 10 array with 700 microns height and 200 by 200 microns base sizepyramid shaped needles prepared from carboxymethyl cellulose solution.The solution is poured, centrifuged and the solvent is removed by airdrying to produce the array. In another example, an array made frompolyvinyl pyrrolidinone is used. In another embodiment, a hyaluronicacid based dissolvable microneedle array implant is purchased fromMicropoint Technologies and is used to prepare cavities.

In another embodiment, a 3 by 3 hollow needle array from MicropointTechnologies Pte Ltd, Singapore is used (referred as 33 MP, FIG. 22B).The hub has a square needle shape with height of 1000 microns,rectangular base 300×300 microns, inner diameter 150 microns, and needlepitch 700-1000 microns and needle's center-to-center spacing is 0.63 mm.Hollow Microneedle Hub (33 MP) with a Luer-slip female hub which can beconnected to syringe with injectable fluid. The needles of thismicroarray are hollow with a common reservoir for injectable fluid. Theinjectable fluid can be connected to a syringe with injectable liquidcomposition. The liquid from the syringe is transferred to the arrayreservoir via Luer-slip connector to the hollow needles which are usedto deposit the liquid where it is needed. Generally, all 9 needlesdeposit liquid from the syringe at the same rate. The height of themicroneedles is 1,000 microns and the needle's center-to-center spacingis 0.63 mm. The internal diameter of the microneedles is 150 microns. Insome embodiments, the array was used on bovine pericardium or sheepdermal tissue or on gelatin gel (transparent tissue like model material)as an experimental material to create porosity. The array is used tocreate 3 by 3 array holes in the tissue with a cavity depth of 1000microns and cavity diameter is same as external diameter/shape of theneedle. The external shape of 33 MP needle is rectangular pyramid whichis a shape of cavity it creates. A PLGA polymer solution with coumarinis used to fill the cavity after its creation. Since the cavity createdhas pyramid like shape, the in situ formed implant has pyramid likeshape.

In some situations, the hollow needle or hollow microneedle array usedto create cavity may cut the tissue (tissue coring) and the cut tissuemay occupy the space inside the needle cavity/hollow space.

To prevent the tissue penetration inside the needle cavity, certainmodifications may be made to create clean well defined space or cavityinside the tissue. In one illustrative embodiment, a 3 by 3 hollowneedle array (33 MP) needles are dip coated using 30 percentcarboxymethyl cellulose solution and air dried. This forms a thin watersoluble film on the outer surface of the needle surface including thehollow needle opening. The coated needle array is then inserted in thetissue. The coating on the needle opening pushes the tissue away fromthe needle and prevents it getting into hollow portion of the cavity.The injectable composition such as polymer solution or fibrin glue orprecursor of crosslinkable composition is then deposited in the hollowneedle cavity. Upon physical and chemical transformation of thecomposition in the cavity, and dissolution of poly carboxymethylcellulose membrane, the needle of the array can be withdrawn from thetissue leaving behind the deposited composition in the artificial cavitycreated by the hollow needle. The water soluble membrane serves astemporary barrier for tissue to enter in the needle cavity and itsdissolution enables withdrawal of the needle without pulling theinjectable composition from the tissue.

FIG. 5 shows partial schematic representation of creation of cavityusing coated hollow needle of microarray and filling the cavity withinjectable composition. 5001 denotes a hollow microneedle of array suchas 33 MP array. The tip of 5001 needle array is coated with waterdissolvable coating or removable coating (5002). The applied coating andits thickness does not affect sharpness of the coating. The coatedneedle is inserted in the skin tissue (1001 and 1002). The coatingprevents insertion of tissue and other material in the hollow spaceinside the needle. The hollow space in the needle inserted in the tissue(5004) is then filled with injectable composition such as fibrinsealant, DuraSeal sealant precursors or biodegradable polymer solutionin water miscible biocompatible solvent (5005). The water in the tissueor components in the injectable material dissolve the coating 5003 whichenables removal of the needle from the tissue without obstruction fromthe coating material. The injectable composition may undergo physical orchemical changes forming solid implant 5006. The injectable compositionmay undergo physical or chemical changes. Coating material used has athickness of 10 microns or higher and preferably 10-1000 microns. It maybe dissolvable in water under physiological conditions such that pH 7.4,temperature 37 degree C. The coating is strong enough to prevent tissuefrom inserting into hollow portion of needle cavity. The preferredmaterial must be biocompatible and biodegradable. The preferred coatingmaterial may include but not limited to are: polycarboxymethylcellulose, polyvinyl pyrolidonone, polyvinyl alcohol, polylactones orpolyhydroxyacids such as PLGA, polycaprolactone and the like.Hydrophobic materials may need to be treated with biocompatible watermiscible solvents like DMSO, NMP, PEDM, PEG to dissolve the coatingprior to injecting the injectable composition. For example, the coatingmay be treated with 0.1 ml NMP to remove from the tip of needle. In somecases, the “array in array” device described in FIGS. 6A, 6B and 15, maybe inserted in the tissue in a closed position (plunger array completelyinserted inside base array, FIG. 15F). This can also prevent tissueentering into needle cavity space. The plunger array is withdrawn thuscreating a space/cavity inside the base array needle which can be usedto fill with injectable composition.

The needles used in hollow microneedle array are preferably non-coringin nature. Generally, non-coring needles are specially designed tominimize coring action during skin tissue insertion. Becton Dickinsoncompany sells Huber trademarked non-coring needles. The designs used inHuber needle may be preferentially used. Huber Needles feature adeflected point (the tip is raised above the centerline to minimizecontact with tissue or media) which eliminates the potential to “core” atissue during insertion process.

In another embodiment, instead of using the membrane coating, the hollowneedle cavity is first filled with tissue dissolvable compositions suchas carboxymethyl cellulose, low melting water soluble polymers like PEGand its derivatives, Pluronics, sugar based compositions, or ice or PBSsolution that is in frozen condition and the like in the hollow cavityand inserted in the frozen state. Polymers like PEG molecular weight2000 to 35000 g/mole or Pluronics and Tetronics are low melting (meltingpoint below 60-70 degree C.) polymers with high water solubility. Suchpolymers may be melted first and then infused in the hollow portion ofthe needle cavity and cooled. The solid polymer formed in the cavityprevents tissue accumulation in the cavity, but is dissolved away in thebody creating a space for filling the injectable composition. The use offrozen water (ice), frozen saline solution or frozen PBS (pH 7.4, 20 mM)may be used in frozen state in place of low melting polymers. Materialsused in dissolvable microneedle array in dry form may also be used.These include but not limited to various sugars, polyvinyl alcohol,carboxymethyl cellulose, dextran, polyvinyl pyrrolidinone and the like.The needles with dissolvable composition are then inserted inside thecavity. With the dissolvable component in the needle, the needletemporarily becomes solid and does not stay hollow. The space occupiedby the dissolvable composition does not permit the tissue to enter inthe hollow space of needle cavity during insertion process. Uponinsertion, the dissolvable composition such as ice or sugar or watersoluble polymer dissolves in the tissue, creating space or cavity forinjectable composition. The dissolved components may also be suctionedoff or aspirated if needed to accelerate the cavity creation process.The injectable composition is then added in the space created by thedissolved composition which conforms to needle cavity shape andtransforms into solid or gel like microimplant. The hollow needle isthen withdrawn from the tissue, leaving behind the formed implant insidethe artificial cavity.

In another illustrative embodiment, cavity creation in the tissue andfilling is done at the same time. A polymer solution is first filled inthe syringe and the syringe is attached to the array via Luer-slipfemale hub. The 3 by 3 array (33 MP) as described above is then insertedin the tissue at full depth (1000 microns) and pulled back about 5 to 95percent (50 to 950 microns in case of 33 MP), preferably 5 to 95 percentand most preferably 20 to 80 percent. Upon pulling back, the empty spacecreated in pulling the needle is then filled with the injectablecomposition fluid from the syringe. The syringe is pressed to inject theinjectable composition such as polymer solution. The polymer solutionoccupies the cavity and the excess solution is oozed or transferred onthe tissue surface. The array is removed, the excess solution from thetissue is wiped off and polymer solution is allowed to precipitate inthe cavity. The NMP or DMSO which is used as water miscible organicpolymer solvent is dispersed by the tissue and which leads toprecipitation of the polymer in the cavity. The deposited polymerentraps the drug. If a volatile solvent such as acetone is used, then acombination of evaporation and tissue dispersion in any proportion maybe used to precipitate/cast the polymer in the artificial cavitiescreated. By choosing variables such as needle height; needle type(hollow or solid); needle shape; needle internal diameter; needleexternal diameter; spacing between each needle in the array; needlematerial type; number of needles per array; number of array insertionpoints and the like, many types of porosities/cavities with differentsize, shape and depth and number of cavities can be created for a givenmedical need. For example, height of the array needle may range from 5microns to 3500 microns, preferably, 10 microns to 2500 microns, evenmore preferably 20 microns to 1300 microns. The number of needles perarray may be greater than 3 or 4 or 5 or 6 and may range from 3 to10000, preferably 4 to 2000. The needle shape may be cylindrical orconical or pyramidal or combination thereof with sharp edges for easyinsertion in the tissue. The shape of the needle also could be, straightobelisk, negative-beveled obelisk, cylindrical, pyramidal, conical,trigonal, tetragonal, pentagonal, hexagonal, pyramidal, irregular andthe like or combinations thereof. The shape of needle may be symmetricalor non-symmetrical. Preferably needle should have sharp edges for easeof insertion. Biocompatible and/or biodegradable lubricants such asvitamin E, silicone oil, coconut oil, mineral oil, oleic acid, liquidpolymers like polyethylene glycol molecular weight 400 to 1000,polycaprolactone (molecular weight up to 1000), glycerol, detergentsolutions like Tween 40 or Tween 80, hyaluronic acid and the like may beapplied on needle surface and/or tissues to lubricate and for ease ofinsertion during cavity creation. The array used may be repeatedlyinserted to create additional holes or cavities. The array could beinserted 2, 3, 4, 5, 6 or more times or could be used up to 2-1000times, preferably 2 to 20 times to create more number of cavities in thetissue. It may be inserted at the same location to stabilize the alreadycreated cavity for 2 or more number of times or it may be inserted at adistance lager than the previous hole created. The distance between eacharray insertion may vary from 1 micron to 10 mm, preferably 2 to 3500microns, even more preferably 10 to 3000 microns. The needle used in thearray may be hollow or solid. If hollow, it may have two, three, four ormore lumens to deposit two or more different injectable compositions atthe same time. The average needle diameter may vary form 5 microns to3500 microns, preferably 10 microns to 2000 microns. The needle arraymaterials may be selected from but not limited to metallic, ceramic,glass, polymeric, silicon, solidified aqueous solutions such as ice(frozen, at temperature below zero degree C. and used at temperaturebelow its melting point). The metallic materials used include but arenot limited to: iron, copper, magnesium, zinc, stainless steel,titanium, brass, silver, gold or their alloys and the like. The commonlyused polymers or plastic materials include but not limited topolyurethane (PU), polypropylene (PP), polyethylene (PE), polystyrene(PS), poly(methyl methacrylate) (PMMA), polycarbonate (PS), liquidcrystal polymer (LCP), and the like. The microneedle implant arrays madeusing dissolvable compositions known in the art may also be used (B.Bediz et al., “Dissolvable Microneedle Arrays for Intradermal Deliveryof Biologics: Fabrication and Application” Pharm Res., volume 31(1),page 117-135, 2014, cited herein for reference only). Rapidlydissolvable materials are preferred in some applications. Thedissolvable microneedles such as described by B. Bediz et al. may beused to create micropores in the tissue. The advantage of such arrays isthat the needles dissolve away upon insertion leaving behind the emptyspace or cavity which can be filled by injectable therapeuticcompositions. Alternatively, surface of such dissolvable microneedlesmay be coated with injectable compositions like PLGA solution in NMP orPEG and then inserted in the tissue. The coated solution is carried awayby the needle in the tissue. As the needle dissolves in the cavity, theaqueous environment precipitates the polymer and entraps the drug insidethe polymer solution. The drug is released by the polymer in a sustainedmanner. Alternatively, as described before, dissolvable microneedlearray may be applied through the polymer solution layer on the tissue asdescribed before. Many dissolvable materials can be used which includebut not limited to: sugars (fructose, trehalose, and raffinose) andpolymeric materials included but not limited to: hyaluronic acid,polyvinyl alcohol, polyethylene glycol and its copolymers,polyvinylpyrrolidone, carboxy methylcellulose, hydroxypropylmethylcellulose, sodium alginate and the like. When using the array withpolymer solution, the array material must be a non-solvent for thesolvent used in injectable composition. Materials like sugars baseddissolvable microarray may be unsuitable because they may get dissolvedin solvent like NMP or DMSO. A list of solvents for polymers and otherchemicals can be found out from chemistry literature, chemistry handbookand polymer handbook.

Mechanical drilling may also be used in some situations, especially insome situations where hard materials like bone, skull and nails areinvolved. In one illustrative embodiment, a 1/64 inch size micro drillbit is used on a human nail (obtained from a human cadaver). 4 cavities,100 micron dip, are created on the nail surface, with spacing betweenthe cavities around 1000 microns each. A PLGA solution in acetone orethyl acetate (10 percent polymer concentration) and 10 percentTerbinafine hydrochloride (relative to polymer plus drug weight,antifungal drug) is applied. The solvent is allowed to evaporate leavingbehind the polymer and drug in the cavity and thus forming a 2 by 2microimplant array. The release of Terbinafine hydrochloride ismonitored over a period of 6 weeks in PBS at 37 degree C. In anotherembodiment, part of human nail is cut. 4 cavities in 2 by 2 array formatare created using a syringe needle (average cavity diameter around 700microns). A PLGA based polymer solution with D and C violet as anillustrative colorant and terbinafine hydrochloride as exemplaryantifungal drug is then used to fill in the cavity. FIG. 16A shows aphotographic image of part of human nail with artificially createdcavities. FIG. 16B shows the cavities (as depicted in FIG. 15A) filledwith PLGA based biodegradable composition with D and C violet ascolorant. FIG. 16C shows in vitro terbinafine hydrochloride (anantifungal drug suitable for treatment of fungal nail infection) releaseprofile from PLGA based experimental composition released from the arrayformed inside the nail.

In some embodiments, a syringe needle is used (an illustrative tool) tocreate cavities manually in the tissue. Approximately 15 mm by 15 mm drypericardium tissue was used to make cavities by hand in 4 by 4 arrayformat. A 24 gauge needle was used to core approximately 1 mm dip cavityby hand in the tissue. Total 4 cavities were made, 2 mm apart from eachother, along the length of the tissue to make one row of cavities. Totalfour rows were made, 2 mm part to make a 4 by 4 cavity array where eachcavity is separated by 2 mm. FIG. 8D shows a pericardial tissue withcavities in 4 by 4 array format as an illustration. Manual method forcavity creation can be useful but may not be preferred where largenumber of cavities are needed. Variables like the size of cavity, numberof cavities made, distance between each cavity, needle size used, depthof cavity can be varied to obtain a suitable array structure. In anotherillustrative example, a 4 by 4 array was created manually by inserting24-gauge needle at 100-300 micron depth in the human nail. FIG. 16Ashows 2 by 2 array cavities created in the nail.

Oscillating needle such as tattoo needle may be used to create cavitiesin the tissue or skin. A commercial tattoo machine as described in therelated application (U.S. Pat. No. 9,072,678, cited herein for referenceonly) is used. The 500-1000 micron size tattoo needle is used and theoscillation frequency was 10 to 12000 oscillations per minute. About 1square centimeter area was treated with the tattoo needle for 1 minute.The pores created by the tattoo machine repeated needle insertion wereused to fill the injectable composition such PLGA solutions with thedrug as described before. Alternatively, polymer solution is firstapplied on the tissue surface to form a solution layer and then thetattoo needle is used to create holes through the solution. The needlegoes in and out of the tissue surface and carries the solution with itinside the cavity created. The deposited solution inside the cavity isprecipitated by the tissue fluids and the precipitated polymer releasesthe drug in a sustained manner. Additional information about tattoobased methods can be found on related applications cited herein forreference only. In one illustrative embodiment (Examples 6D, 17),bupivacaine base releasing microimplants array was prepared usingoscillating needle to create porosity and infuse polymer solution in theporosity. Briefly 172 mg PLGA polymer (PDLG 5002) is dissolved in 1.75ml DMSO. 0.75 ml of polymer solution and 23 mg of bupivacaine are mixed,and the solution is applied on the glutaraldehyde fixed bovinepericardium tissue. A commercial tattoo machine needle (permanent makeupmachine needle) is used to create the porosity and drive the solutioninside the tissue. The needle is moved on one square centimeter diameterarea. The machine needle oscillated at 6000 times per minute. Afterabout two minutes, the oscillating needle machine is stopped and excessbupivacaine solution is wiped off from the tissue surface. Care is takento ensure that no polymer sample is precipitated on the tissue surface.A control sample is prepared/tattooed using identical conditions whereonly polymer solution in DMSO without drug is used for infusion. Thetreated areas (bupivacaine treated and polymer treated control) were cutfrom the tissue and were subjected to drug release in PBS at 37 degreeC. for several days. The concentration of bupivacaine in the elutedsamples is monitored using UV spectrophotometer. A bupivacaine releaseprofile elution curve is shown in FIG. 13 along with polymer. Thepolymer solution was successfully infused by the oscillating needle. TheDMSO is dissipated in the tissue leaving behind PLGA polymer along withhydrophobic bupivacaine. The release from the precipitated polymer isshown in FIG. 13. It is clear from the FIG. 13 that the sustainedrelease of bupivacaine base is possible for several days. By changingtype of polymer used, drug concentration in the polymer, polymermolecular weight, number of microimplants, implant shape and the like, asuitable drug release profile may be designed for a given medicalcondition.

A microneedle fractional radiofrequency (RF) device can also be used tocreate artificial cavities in the tissue. Such devices are commerciallyavailable from Lutronic Corporation or Cryomed Corporation. (LutronicCorporation, Lutronic, North America, Burlington; Cryomed Corporation,Sydney, Australia). The array device available from suppliers as aboveor other vendors insert an array of microneedles (5 by 5 array as anexample) in the skin tissue. Upon insertion of microneedle array in theskin tissue at a controlled depth (300 microns to 3.5 mm as an example),the needles are supplied with controlled RF power which is transmittedto the surrounding tissue causing controlled denaturation of the tissuesurrounding the needles and also forms a micro cavity in the areasurrounding the needle. The shape and size of the cavity formedgenerally depends on the variables like total RF power applied via arrayneedles, needle depth, needle size and shape and the like.

Additional information about the device and its use can be found inByalekere S. C. et al. (J Cutan. Aesthet Surg., Volume 7(2), Page 93-97(2014)) and references therein; cited herein for reference only. Thistype of method uses combination of both methods such as tissuedisplacement (during needle insertion) and tissue destruction (applyingRF power to destroy or denature tissue) to create cavities.

In one embodiment, the needle penetration is controlled by the using thespacers. For example, a 500 micron thick polymer adhesive film is firstapplied on the tissue and a device like 33 MP is used through the film.Because the polymer adhesive film has a thickness of 500 microns, the 33MP device needles with 1000 microns needle depth can only penetrateabout 500 microns in the tissue. By changing the thickness of the spaceror polymer film, the depth of penetration can be controlled. In anotherembodiment, the array needles are placed in a “tube in a tube” likedevice wherein inner tube can be moved out of outer tube using a screwlike movement or using a shaft of a linear motor. The needles are placedon proximal end of inner tube and it is moved out of outer tube atprecise length. The inner tube array needles come out of outer tube atpredetermined length (500 microns as an example). When outer tube ispressed against the skin tissue, the needles up to 500 microns go firstinto the tissue but cannot go further because of the outer tube preventsit from going it further. Thus the “tube in tube” arrangement of needlesand movement of inner and outer tubes can be used to control the depthof penetration. A NuCell skin solution (a Dermapen like device,purchased from Amazon Inc. uses “tube in tube” like arrangement tocontrol the depth of needle penetration. A 36 needle cartilage isinserted in the device and connected via Bayonet Coupling mechanism tothe device and the outer tube of the device is rotated clockwise oranticlockwise to adjust the needle exposure or penetration depth. Thedevice has a gauge to adjust the penetration depth from 250 microns to 2mm. In another embodiment, a 36 pin Needle Cartridges for Derma Pen(micro-needling skin dermabrasion medical device). The Dermapen is anautomated micro-needling device, with a disposable needle tip cartridge,that uses 9-42 microneedles array to vertically stamp the skin at highspeed. The stamping action of the Dermapen's vertical tip createsmicro-cavities in the skin. In one embodiment, A NuCell skin solution (aDermapen like device, purchased from Amazon Inc.) is used to createcavities. The machine is fitted with 36 needle sterile cartilage(Purchased from Amazon, UPC code 601913872222) via Bayonet Couplingmechanism and the needle length is adjusted to 500 microns. The lengthadjustments protrude 36 needles out of the machine at the length of 500microns. An outer plastic tube on the cartilage prevents the needle togo beyond 500 microns inside the tissue. The machine can be adjusted topenetrate from 250 microns to 2 mm in the skin tissue. The 36 needleswith 500 microns penetration depth are stamped at the same location 10times to create 36 artificial cavities in approximate one centimetersquare circular area. In another embodiment, a 9 pin cartilage is usedto create 9 cavities at 250 micron depth in the sheep skin tissue.

In some embodiments, it is envisioned that only some of the needles areprogrammed to penetrate the tissue out of several available needles. A10 by 10 array needle containing 100 needles is used as an illustration.The mechanism wherein only one, or two or 3 or 4 or 10 or 20 needles cancome out the array and used for injection. This type ofmechanism/arrangement can help to control total drug dose given for agiven surgical tissue or skin site. The pattern created by use ofprogrammed insertion may be used to code certain information like typeof drug used, its dose, date and time and the like.

In some cases, computer controlled machines may be used to createporosity and to fill cavities. Such machines may deploy microimplantarray based devices as described in this invention. Exemplary machinessuch as da Vinci® Surgical System (Intuitive Surgical, Inc. Sunnyvale,Calif.) or other MIS surgical based instruments known in the surgicalart may be preferentially used to deploy microimplant array baseddevices and compositions described in this invention. The advantage ofrobotic machine based cavity creation is that more closely spacecavities of precise depth and diameter can be made in a reproduciblefashion for a given medical need.

Another preferred way to prepare porosity in the tissue is to use laserbased methods already practiced in the medicine. For example,ophthalmologists use laser based systems to correct nearsighted visioncorrection generally referred as LASIK procedure. Laser based tools usedin LASIK surgery may be used to drill holes or create cavities in thelive or bioprosthesis tissue. Ablative fractional laser therapy is usedto treat variety of skin conditions including drug delivery. In oneliterature reference, cited herein for reference only, E. H. Tudor etal. (Lasers in Surgery and Medicine, volume 46, Page 281, 2014 andreferences therein) describe the use of erbium yttrium aluminum garnetlaser (Er:YAG; 2940 nm) to create several size micro-channels or holesor cavities in the pig skin. The authors were able to create conicalshaped channels/holes with ablation width 22 microns to 488 microns and16 microns to 1348 microns ablation depth (E. H. Taudorf et al., Table 2in the reference). The shape of cavities created can be seen inhistology (E. H. Taudorf et al., FIG. 1 in the reference). The authorsmodified various instrument parameters (laser power, beam width, laserpulse rate, number of stacks to achieve to control cavity diameter anddepth. Many types of ablative fractional lasers (AFXL) instruments arecommercially available and are used in modern medicine practice. Thesemachines could be used to create micro-porosity in the live tissue,preferably in the skin tissue or in bioprosthesis tissue. Erbium:yttriumaluminum garnet laser (Er:YAG; wavelength 2940 nm), carbon dioxide laser(CO2; wavelength 10600 nm), yttrium scandium gallium garnet laser (YSGG;wavelength 2970 nm) and the like are some of the most commonly usedlasers in medicine practice. These laser instruments emit laser light ininfrared range and target water in the tissue. The energy for the laserbeam is absorbed by the water present in extracellular matrix intissues, which leads to evaporation of water and surrounding tissueproducing a void or channel or cavity in the tissue. Variables such aslaser wavelength, laser spot size, laser power level, laser pulseduration, laser pulse repetition rates, number of stacked pulses and thelike can be controlled to obtain cavities with various shapes and sizesin the tissue. Those skilled in the art will understand that manyvariations are possible and ultimate parameters will depend on desiredcavity diameter/size, shape and depth. In general, lasers used ininfrared wavelength potentially may create local thermal injury. UVbased laser on the other hand, do not create thermal injury andgenerally provide a clean cut. However, UV radiation can penetrate onlyat a small depth and generally useful of creating shallow cavities withsmall size of diameter. UV laser can create much smaller diameter holesthan infrared laser and therefore may be preferred where small diameterchannels (1 to 100 microns) are desired. In modern laser basedinstruments, generally the machine parameters are controlled by thecomputer software. The focused laser beam is scanned across desiredtissue area. The laser beam diameter is a function of optics used in theinstrument as well as wavelength. UV based laser can be focused on amuch smaller diameter as compared to infrared or visible light lasers.The laser energy delivered to the tissue is a function of residence timeof laser beam on the tissue surface, total laser power and repetition oflaser pulse frequency and the like. Depending on porosity desired, laserwavelength, laser power, laser pulse frequency and other instrumentparameters may be varied to obtain a suitable porosity level and cavitysize.

There are many methods that can be used to generate porosity in thetissue and preferred methods are discussed above. Those skilled in artunderstand that porosity preparation methods known in the art or yet tobe discovered may also be used. Water jet drilling based methods,ultrasonic energy based methods, particle bombardment based methods andthe like could also be used in preparing artificial porosity in thetissue. Among these, porosity preparation using laser based methods,oscillating needle, mechanical drilling and microneedle array basedmethods are most preferred. Among the microarray based methods, use ofdissolvable microarray or hollow metal microneedle array is mostpreferred.

The artificial cavities created by methods discussed above may createpores or cavities with various shapes, volumes and sizes. The averagediameter of cavity prepared may range from 0.5 microns to 3500 microns,preferably 1 micron to 2500 microns, even more preferably 10 microns to2000 microns. The depth of cavity prepared may range from 1 micron to5000 microns, preferably 5 microns to 3000 microns, even more preferably10 microns to 2000 microns. The shape of the cavity created may rangefrom straight obelisk, negative-beveled obelisk, cylindrical, pyramidal,conical, trigonal, tetragonal, pentagonal, hexagonal, pyramidal,irregular and the like or combinations thereof. The distance betweeneach cavity may range from 1 micron to 10 mm, preferably 3 microns to3500 microns, even more preferably 5 microns to 2000 microns. The volumeof each cavity may range from 1×10E-12 to 0.05 ml, preferably 1×10E-10to 0.03 ml, even more preferably 1×10E-10 to 0.01 ml. Total numberartificial pores or cavities created may be greater than 4 per squarecentimeters or may range from 4 to 6000 per square centimeter. Totalnumber artificial pores or cavities created may be greater than 3 or mayrange from 3 to 20000, preferably 3 to 15000, most preferably 3 to 10000per treatment area. The microimplants created by in situ casting ofinjectable compositions in the cavities described above will havesimilar dimensions and shapes similar to the cavities created as above.Preformed implants of similar size and shape as above can be inserted incavities to create microimplant array. The preformed implants may beporous in nature.

When using microneedle array for creating porosity, generally theneedles are inserted at 90 degree angle (perpendicular) to the surface.In some applications, that angle may be shifted and could be changed to30 to 80 degrees, especially implanting prefabricated implants asdescribed in this invention. For example, needle of microneedles may bespecially designed such that during tissue insertion, the needles maypenetrate at 45 degree or 30 degrees relative to the skin surface. Someof the devices described in this may have microneedles that can insertinside the tissue at 20-80 degree angle, preferably 30-60 degree angle.

When using microneedle array for creating porosity, generally the use ofapplicator to apply the array on the skin/tissue is desirable. Theapplicator is specifically designed to apply definite force in the rangeof 15 N/CM². This enables better insertion of array needles in thetissue. Generally commercial suppliers of array materials can provide anapplicator for obtaining highly reproducible results while using theirarray product. Human hand when used properly can be used without theapplicator if trained properly. A specific applicator may also bedesigned and used for a given microneedle array. A robotic machine maybe programmed to exert force in the range of 5 to 30 N/CM², preferably10 to 20 N/CM² and even more preferablyl 5N/CM² force. Similar amount offorce may be used in inserting AIA device needles.

The porosity creation as described above may involve either tissuedisplacement or tissue destruction or combination of both. Use ofdissolvable microneedle array generally involves tissue displacement andthis type of porosity generally does not remove or destroy the tissue.Methods such as micro-drilling in bone tissue or infrared laser burningof tissue and the like, physically remove the tissue from its existingspace and create a space or cavity. The choice of cavity creation willdepend upon the clinical need and desired outcome. In general, methodsusing non-destructive removal of tissue such as use of dissolvablemicroarray needle array are preferred or use of specialized devices suchas “array in an array” type described in this invention may also beused. Methods that displace the tissue rather than destroy the tissuealso are most preferred.

Infusion of Injectable Compositions in the Artificial Porosity

Methods of Filling the Artificial Cavities

The artificial cavities created as described in previous sections arefilled with injectable compositions that can provide sustained releaseof drugs. The filling materials are generally in a fluid state or in theliquid state and have ability to flow in the tissue cavity or porosity.The liquid composition may be a low to high viscosity. Viscousinjectable liquids, preferably with low viscosity (1 to 500 Centipoise)to medium viscosity (500 to 10000 Centipoise) are preferred. The liquidcomposition injected may be solution, emulsion or suspension orcombination thereof comprising a carrier matrix and bioactive compoundor drug or may comprise live cells. The fluid composition may be appliedon the cavities and it is pulled into cavity via gravity. If needed,additional pressure/force may be applied on liquid composition to forcethe composition in the cavity. The pressure may be applied using a gasor liquid means. For example, the liquid composition may be firstapplied on top of the porous area and the area is enclosed using anenclosure device. The enclosure device is connected to a gas line suchas carbon dioxide, oxygen or other biocompatible gas. The enclosure isthen filled with gas and gas pressure is increased in the enclosure. Thepressurized gas transfers its pressure on the injectable composition ontop of cavities which helps the composition to enter in the cavities andfill the cavities with the composition. In one embodiment, a pressurizedgas stream coming out from a 19-gauge syringe needle is used to drivethe injectable composition in the artificial cavities. Instead of gas,biocompatible fluids such as PBS or other biological biocompatibleaqueous buffers may be used to apply pressure and inject thecompositions. Biological fluids that may be used include but not limitedto are: phosphate buffer pH 7.2, triethanol amine buffer pH 7.2, HEPESbuffer pH 7.2 and the like. Care is taken to ensure that the liquid useddoes not affect the injectable composition or does not prematurelyprecipitates before going in the cavities. In some case, injectablecomposition may be sprayed or atomized and the fine droplets are forcedinto artificial cavities. Other energy based methods such as use ofmagnetic force, ultrasonic waves, laser radiation and the like may alsobe used in filling injectable compositions in the cavities.Alternatively, injectable compositions may be filled using syringe likedevice and injected in the cavities with or without pressure. Thoseskilled in the art understand many methods can be used in assisting infilling the cavities; the ultimate choice will depend on the injectablecompositions, its viscosity and other injection parameters. The cavitiesmay be filled partially (1 percent to 99 percent, preferably 10 to 90percent of cavity volume occupied by the composition) or completely withinjectable compositions. It is preferred that at least 10 percent orhigher cavity space is occupied by the composition. It is not necessaryto fill all the cavities. Depending on the desired clinical outcome,drug concentration, 1 to 99 percent, preferably 10-90 percent ofavailable cavities may be filled with the injectable composition.

The injectable compositions may be filled in the cavity with one, two,three, four or more layers and each layer may have a differentdrug/cells or biodegradable polymers or combinations thereof. Two ormore injectable compositions may be filled in layers to form a multilayered implant. Each layer may have a drug or cell or visualizationagent. The multilayer approach may be used in some cases to achieve adesired release rate. In a three layered implant the top and bottomlayer may have biodegradable polymer without drug and the middle layerhas a drug. The drug diffuses through the top and bottom layers andaffects its release profile. Alternatively, top and/or bottom layers maycontain a visualization agent such as fluorescent or colored compound. Aprefabricated multi-layered implant may be also inserted in the cavityto form an array.

Microfluidics is the science of manipulating fluids at micron andsubmicron levels. Many microfluidics devices are available commerciallythat can be used for variety of scientific and technical applications.Please refer to review of microfluidics devices by L. Y. Yeo et al.(Small, 2011, Volume 7(1), Page 12-48, (2011) and R. G. Willaert et al.(Fermentation, Volume 1, Page 38-78 (2015) and references therein; citedherein for reference only for additional information. In one embodiment,a glass or silicone rubber based microfluidic array with 9 fluidchannels is designed and each output of the channel is fed to amicroneedle of 3 by 3 array using a specially designed connector. Thisway, small volumes of injectable compositions can be used to inject viamicrofluidic array into the artificial cavities. Commercial firms likeuFluidix (Toronto, Canada); Fluigent Inc. (Lowell, Mass.) can also helpto design and make desired microfluidic device for a given use.Injectable compositions comprising drug/cell encapsulated microparticles

A partial and schematic representation of in situ generated drugdelivery array comprising drug encapsulated microparticles is shown inFIG. 7. A partial schematic of skin tissue is represented by epidermis(1001) and dermis (1002) layers. Artificial porosity is generated in theepidermis and/or dermis layer (7001). Conical cavities (7001) formed inthe skin tissue are schematically shown. The cavities (7001) are filledwith fluid injectable drug delivery composition comprisingmicroparticles encapsulated/coated with drugs (7002). The drug isreleased from the microparticles in the cavity and in the surroundingtissue. Example 14C provides an illustrative method for infusingrifampin loaded microspheres in the skin tissue. FIG. 14 shows a drugrelease profile of rifampin encapsulated microspheres from microimplantsarray formed in the tissue using a microneedle array. The artificialcavities are formed through the rifampin microspheres suspension inglycerol on the sheep skin tissue surface by the microneedle array. Thearray needles are pressed on the tissue through the suspension. As theneedle penetrates the tissue and form a cavity, the suspension iscarried along with it. The glycerol is dissipated in the tissue leavingbehind rifampin microspheres in the artificial cavities. Microsphereswithout rifampin were also incorporated in the tissue and used as acontrol. Rifampin release profile from the tissue and from the control(lower curve, solid circles) is shown. As expected the rifampinmicrospheres showed a sustained release of rifampin in the tissue andcontrol microspheres did not show rifampin release (solid circles,bottom curve).

The composition may be biodegradable or biostable microparticlesencapsulated or coated with drugs or bioactive compounds. In oneillustrative embodiment, PLGA microspheres (average size 1-100 microns)containing 10 percent rifampin as a model drug is suspended in aqueousmedium such as PBS or in glycerol and then filled inside the cavity. Itis understood that the cavity size must accommodate the microparticlesize distribution. For example, in the illustrative example, theparticle size is in the range of 1-100 microns. The cavity size must beat least 5 percent larger than the largest size of microparticlespresent in the injectable composition. In this case, the averagediameter of cavity must be 105 microns (5 percent of 100) or higherpreferably 120 microns (20 percent higher) or higher even morepreferably in the range of 105-400 microns (5 to 400 percent higher).This will enable to fill the cavity with microparticles. Themicroparticles will release the drug to the surrounding tissue forsystemic or local therapeutic effect. Please refer to the U.S. Pat. No.9,072,678, and references therein, cited herein for reference only; forpreparation of microparticles with drug. It is preferred that fluidcomposition comprises visualization agent which helps to see thecomposition during cavity filling operation. The composition can bevisualized by aided or unaided human eye or with the help of medicalimaging equipment such as x-ray machine (for radio-opaque composition)or magnetic resonance imaging machine (for paramagnetic composition) orultrasound imaging machine. The preferred visualization agent is coloredor fluorescent in nature. In some preferred embodiments, thevisualization agent is incorporated in the microparticles. Forpreparation of colored microparticles, please refer the U.S. Pat. No.9,072,678, and references therein. The cited patent provides variouscompositions and methods for obtaining colored microparticles. It ispreferred that drug particles are encapsulated in the microparticles,preferably in biodegradable microparticles or microspheres. Many methodsare known in the art to make biodegradable particles. U.S. Pat. No.9,072,678, and references therein. Some preferred methods are given inU.S. Pat. No. 9,072,678, Table 2. One embodiment teaches to encapsulateF D and C dye and drug in a same microparticle. Another embodimentteaches making colored and drug coated microparticles separately andusing the mixture of these two particles in any proportion to obtainsuitable drug loading and color depth. Drug and color compound loadingin particles is controlled to obtain a suitable drug release rate orcolor depth. The drug/color compound loading in the biodegradablepolymer used for encapsulation may range from 1 to 50 percent relativeto the weight of polymer, preferably from 5 to 30 percent. Coloredmicroparticles may also be obtained by staining the biodegradablepolymer particle or may be obtained by encapsulating within the polymer.In one case the drug (rifampin) itself has a mild color and serves asdrug as well as coloring agent. In another embodiment, the drugparticles are stained with staining compound and used withoutencapsulating in the polymeric carrier. Poorly water soluble drugs suchas chlorhexidine, paclitaxel, silver chloride and the like which havewater solubility less than 5 g/100 g water are especially useful forthis application. Microparticles/microspheres and even more preferablywith biodegradable polymer microspheres can be useful for local drugdelivery applications. The definition of biodegradable polymer isincluded in the definition section. The preferred biodegradable polymersused for encapsulation of drug are: polyhydroxy acids, polyester,polylactones, PEG, polytrimethylene carbonate or their copolymers orblends. Hydrogels, biostable or biodegradable polymers could also beused as drug delivery vehicles. Some preferred embodiments providemethods and compositions for preparing hydrogel based drug deliverysystems. The drug particles must be suspended in the liquid medium suchas PBS (pH 7.2). Other aqueous solutions include saline solution; wateralcohol, water glycerine, water PEG, water protein (albumin) mixturesand the like may also be used. Water with biocompatible buffers is apreferred medium. Additives that may be added to the particleformulations may include but not limited to: wetting agent to remove airfrom particle surface; dispersing agent or surfactant to form a stablesuspension; and a liquid medium that maintains the particles in a fluidform. An additive that improves the loading of drug suspension in theneedle may also be used. The type of drug used will depend on themedical condition being treated. The list of drugs is clearly defined inthe definition section including additional list drugs cited in thereference may be used. The list of drugs is not limited to drugsmentioned in the definition sections. Other drugs compounds cited inU.S. Pat. No. 8,067,031 could also be used, cited herein for referenceonly. Biodegradable microspheres/microparticles can be fabricated usingvariety of methods and can be formulated to release drugs at a certainrate (kinetics of drug release). The drug may be released by diffusionand/or biodegradation mechanism or combination of both. The preferredrate of release is a zero order release where a constant or nearlyconstant rate of drug over a long period of time is obtained. Polymermolecular weight, type of polymer used, microparticle size and shape,double or single walled particle, drug loading in the microparticle,porosity of the particles and the like are some of the variables thatcan be used to obtain desired rate of release for a given therapeutic orbioactive compound. If needed combination of two or more microparticlesmay be used to obtain burst release and/or zero order release of drugs.Please refer to U.S. Pat. No. 6,599,627 and cited art and crossreferences therein to make biodegradable microspheres, cited herein forreference only.

There may be other methods known in the art to make biodegradablemicrospheres, such methods could be used or methods yet to be developedcould also be used. By mixing two or more colored particles, preferablyprimary color particles, a desired color shade may be created. Manytypes of biodegradable polymers could be used to make color particles.The list is exhaustive but preferred polymer include polymer, copolymersof polylactones or polyhydroxyacids, and polytrimethylene carbonate.PEG-polylactone PEG-polycarbonate polymers are also preferred. Amonghydrogel polymers, the PEG based crosslinked hydrogels and protein basedhydrogels are preferred carriers for colored substances. In someapplications hydrogels may be preferred because hydrogels in dry statecan form very small size particles and once injected can absorb up to0.1 to 20 times or even more to its original weight water whichincreases their size and therefor are unlikely to move away frominjection site. Hydrogels that absorb 10 to 10000 percent water uponinjection are most preferred. Some embodiments in this inventionillustrate methods to obtain hydrogel microspheres (Example 4 and 5).Such microspheres may be dried or dehydrated and used. PEG basedhydrogels are prepared by crosslinking PEG based macromonomers orcrosslinking reactive precursors.

Methods of preparing biodegradable hydrogels are known in the art(please refer to U.S. Pat. Nos. 5,410,016 and 6,566,406 and referencescited therein, cited herein for reference only) and such methods mayalso be used. Methods described in the cited patents can be used toobtain biodegradable hydrogels with different amount of in vivodegradation time. Methods described in these patents could also beadopted to make hydrogels microspheres. Methods provided in U.S. Pat.No. 6,599,627 and cited art and cross references therein, cited hereinfor reference only may also be used to make colored biodegradablemicrospheres.

In another embodiment, islet encapsulated microspheres are made (Example18) and live cell containing microspheres are used to fill theartificial cavities in the tissue. The microencapsulation matrix used issemipermeable allowing critical nutrients from surrounding tissue andmaintaining cell viability. The matrix also protects the cells fromimmune reaction by preventing diffusion of immunoglobulins. The matrixis also permeable to insulin which is produced for live cells inresponse to glucose concentration in the tissue fluids.

Examples 3-5 show illustrative methods to make drug encapsulatedmicroparticles. Additional methods are given in U.S. Provisionalapplication 62/378,662 filed on Aug. 23, 2016 and its relatedapplications. Methods such as spray drying method, freeze-drying method,melt method can also be used to make encapsulated microparticles.Artisans can understand that many modifications can be done to thesemethods to obtain drug loaded microparticles, preferably microspheresthat have desired size, distribution and drug loading. In addition,compounds such as coloring agent may be added during particlepreparation to obtain a drug loaded microparticle with color. Example 3teaches one illustrative method for obtaining colored and drugencapsulated composition in the same particle. Microparticles with drugsand microparticles with coloring agent can be mixed together to obtain adesirable color as well as release profile. The mixing can be done inany proportion to obtain desirable color and drug loading. Two or morecolored particles may be mixed to obtain a desirable color shade. Oneembodiment teaches the preparation colored hydrogel based composition.Alternatively, many commercial companies/entities provide biodegradablemicrospheres for a given clinical application, such companies may becontracted to provide an encapsulated microparticle composition.Companies like Octopus N.V. Netherlands, Nanomi B.V, Netherlands;Polysciences, Inc. Warrington Pa., Alkermes plc Waltham Mass., RamanncoInc., and the like could be used to make custom based sustained releasemicroparticle compositions, preferably biodegradable microspheres for agiven application.

Injectable Compositions Comprising Polymer Solutions and Drug. In thisinvention, the artificial porosity in the tissue is filled with apolymer solution with or without a drug. Partial and schematicrepresentation of making in situ generated drug delivery array forsustained drug delivery is shown in FIG. 2. A partial schematic of skintissue is represented by epidermis (1001) and dermis (1002) layers.Artificial porosity is generated in the epidermis and/or dermis layer bymany methods known in the art or described in this invention. Conicalcavities (2001) formed in the skin tissue are schematically shown. Thecavities (2001) are then filled with fluid injectable drug deliverycomposition comprising drug/s or bioactive compound/s (2002) andbiodegradable polymer dissolved in a water miscible organicbiocompatible polymer solvent. The fluid composition is converted intosolid or semisolid or hydrogel (2003) by precipitating the polymer andentrapping the drug in the in situ formed solid or gel. The drug isreleased from the solid or gel in the surrounding tissue by diffusionand/or biodegradation or combinations thereof processes. The depositedpolymer entraps the drug which is released in a sustained manner forlocal or systemic therapeutic effect. Both the biostable andbiodegradable polymers can be used to deposit in the cavity, butbiodegradable polymers are preferred. In one illustrative embodiment,PLGA, (polylactide-co-glycolide) (lactide:glycolide (50:50)), molecularweight 10000 to 15000 g/mole, ester endcapped) an exemplary syntheticbiodegradable polymer that is water insoluble is used as a carrier forthe drugs. The polymer is dissolved in n-methyl pyrrolidone (NMP), anillustrative biocompatible water miscible polymer solvent along withcoumarin as a model drug or rifampin as exemplary therapeutic drug andmethylene blue as a colorant. The polymer solution at 10 percent drugloading (relative to polymer plus drug weight) is sterile filtered usingan inert Teflon or polypropylene based syringe filter. The solution isthen applied on the porcine or sheep dermal tissue skin where nine (300micron diameter and 1000 micron height) cavities were created by usinghollow stainless microneedle array (33 MP). The polymer solution isincubated with the cavities for 10 minutes to fill the cavities. In oneembodiment, a nitrogen jet (via glass capillary tube or stainless steelsyringe needle, 20 psi) is used to force the solution inside the cavity.In another embodiment, where cavities are relatively large, a syringeand needle is used to fill each cavity manually. The needle size of thesyringe preferred to be smaller than the cavity size. The excesssolution is wiped off and NMP is allowed to dissipate in the tissue. Thepolymer precipitates in the cavity entrapping the coumarin or rifampin.The presence of rifampin is clearly seen in the precipitated polymer dueto its mild yellow/red color. The precipitated polymer cannot be removedby manually wiping down. The presence of polymer in the tissue is laterconfirmed by conducting histology of treated tissue. The polymerpresence is also confirmed by observing the precipitated polymer withthe necked eye under blue light (coumarin produce green fluorescentlight when observed under blue light, FIG. 8C, 8004). The red color ofrifampin is seen by the necked eye. The treated areas are cut andincubated in 3 ml PBS at 37 degree C. and the fluid is exchanged at 10minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 1day, 2 day, 3 day, 7 day and twice a week up to 30 days. The drugconcentration in the fluid is monitored by UV-VIS spectrophotometer. Adrug release profile is generated and cumulative drug released isplotted against cumulative time. Red color of rifampin and greenfluorescence is only seen in areas where cavities are created and not inother areas. This experiment demonstrates that PLGA polymer array can beformed by making the cavities first and then forming the PLGA implant insitu inside the cavity via in situ precipitation of polymer solution.The formed implant does not require sharp edges or backing material tomake the array implant. In another embodiment, 1 ml of 10% PDLG 5002polymer solution in n-methyl pyrrolidone (NMP) is mixed with 200 mg ofEosin stained MgCO3. The solution/suspension is filled in the syringeand manually injected in the cavities of sheep tissue (cavities weremanually created by syringe needle, 4 by 4 format). The excess solutionis wiped off and the polymer is allowed to precipitate in the cavitiesto make a 4 by 4 microimplant array (8007, FIG. 8E). The biodegradablePLGA based microimplant array (8007) is fluorescent under blue light dueto eosin in the microimplant and its image is shown FIG. 8E.

Example 14B illustrates the creation of PLGA based moxifloxacinreleasing microimplant array in the tissue. The release profile ofMoxifloxacin released from the created array is shown in FIG. 10. Thearray of microcavities were prepared first and then filled with polymersolution comprising PLGA. The solvent diffuses in the tissue and thepolymer encapsulated moxifloxacin releases the drug for several hoursindicating successful encapsulation of drug in the precipitated polymer.Example 14A illustrates the use of direct injection of exemplary PLGApolymer solution in DMSO or NMP with Moxifloxacin as an illustrativedrug and methylene blue as illustrative colorant. The direct injectionwas made using 3 by 3 hollow needle array (33 MP). The array was used tocreate porosity/cavities and inject the polymer solution cavities.Polymer in the artificial cavities is precipitated encapsulating themoxifloxacin. The release of moxifloxacin from the precipitated polymer(PLGA array formed in the tissue) is shown in FIG. 12. The drug isreleased over a period of 200 hours. Example 14C illustrates the use ofpolymer solution layer to form implant array in the tissue. The PLGA andmoxifloxacin solution in DMSO was poured on the tissue first to form aliquid solution layer on the tissue surface. A 3 by 3 hollow microneedlearray (33 MP) was pressed on the tissue via polymer solution layer 15times on different locations on the tissue. As the array needlespenetrate the tissue and they form cavities inside the tissue, thesolution was then carried in the cavities (135 total cavities). Theexcess surface solution was wiped off with a tissue paper. The infusedsolution is converted into precipitated polymer in the artificial porescreated by the microneedles. The release profile of moxifloxacin fromthe formed PLGA implants in the cavity over 10 days is shown FIG. 11.

FIGS. 8A, 8B, 8C, 8D and 8E show representative images of cavitiesformed in tissue and gelatin gel and then filled with polymers with drugand/or visualization agent. A microimplant array is formed in the modeltissue like material (gelatin gel, 8001) and sheep skin tissue (8005) orpericardial tissue (8008). A 3 by 3 array (33 MP) is used to createporosity in transparent gelatin gel (8801) which is then filled withPLGA polymer containing methylene blue as a colorant and/or drug. Anillustrative image of 33 MP array device and attached syringe with bluecolored solution PLGA is shown in FIG. 22B. The precipitated PLGApolymer and its blue color in 3 by 3 microimplant array form (8002) areshown in FIG. 8A. The gelatin gel has blue tint due to leakage ofcolorant in the gel from the PLGA microimplant array. FIG. 8B showsgelatin gel with 3×3 microimplant array made from PLGA polymer solutionand coumarin as fluorescent dye using 33 MP array. The 3 by 3 PLGAimplant array formed in situ which is fluorescent under blue light(8003) is shown in FIG. 8B. A PLGA polymer with coumarin microimplantarray were formed by direct injection in the sheep dermal tissue (8005)using 33 MP array at 3 separate locations is shown in FIG. 8C. Theformed microimplants are fluorescent under blue light (8004). FIG. 8Dshows cavities (8009) created in pericardial tissue (8008) beforeinfusion of injectable composition. FIG. 8E shows sheep skin tissue(8005) infused with 4×4 array (8007). The microarray 8007 is made byinfusing PLGA polymer solution containing magnesium carbonate stainedwith eosin. The implanted microarray 8007 is pictured under blue lightwherein eosin in the array is fluorescent.

In another embodiment, a PEG-PLA block copolymer is dissolved in ethanoland injected inside the cavity. In another embodiment, apolycaprolactone polymer dissolved in dimethyl sulfoxide is used asinjectable polymer solution. Several biodegradable polymers are known inthe art and can be used for sustained delivery. A partial list ofpreferred biodegradable polymers is provided in the definition section.The preferred polymers are synthetic biodegradable polymers whichinclude, but are not limited to, polymers, dendramers, copolymers oroligomers of glycolide, dl-lactide, d-lactide, 1-lactide, caprolactone,dioxanone and trimethylene carbonate; degradable polyurethanes;polyamides; tyrosine-derived polycarbonates, tyrosine-derivedpolyacrylates, polyesters; polypeptides; polyhydroxyacids; polylacticacid; polyglycolic acid; polyanhydrides; and polylactones; polyethyleneglycol-polyhydroxy acid or polyethylene glycol-polylactone copolymers(PEG-PL copolymers); polyvinyl alcohol co-polylactone copolymers areamong the hydrophobic synthetic polymers could also be used. Thesepolymers can be dissolved in biocompatible organic solvents. Eachpolymer used can have its own set of organic and water based solvents.List of solvents that can be used for a given polymer can be found inPolymer Handbook. Alternatively, solubility can be determinedexperimentally prior to using. In general, water miscible solvents aremost preferred. Among these, solvents that can be tolerated by livetissues are mostly preferred. The partial list of solvents and mixturesin any proportions that can be used include but not limited to:tripropionin (triprop), tetraglycol, pyrrolidone-2, ethyl lactate,triacetin, triethylene glycol dimethyl ether (triglyme), glycerolformal, dimethyl sulfoxide, ethylene glycol monoethyl ether acetate,benzyl alcohol, n-methyl pyrrolidone, N-ethyl-2-pyrrolidone, tributyrin,benzyl benzoate, acetone, methyl ethyl ketone, acetic acid, ethanol,isopropanol, diethylene glycol dimethyl ether (Diglyme), ethyl benzoate,dimethyl isosorbide (DMI), polyethylene glycol dimethyl ether,glycofurol, glycerol, ethyl acetate, polyethylene glycol (low molecularweight), 1,3 propane diol, 1,4 butane diol, 1-6-hexane diol,tetrahydrofuran, triethanol amine, water, buffered water solutions withpH ranging from 6 to 8, preferably pH around 7 and their mixture and thelike. If water based solutions are used, it is preferred that thesolutions are osmotically balanced and appropriate pH and buffer tomaintain the pH is used. Among these, polyethylene glycol, ethylbenzoate, polyethylene glycol dimethyl ether (preferred molecular weight500-35000 g/mole, linear or branched), glycofurol, ethanol, dimethylsulfoxide, acetone, water and n-methyl pyrrolidone and their mixtures inany proportion are most preferred. The polymers concentration in thesolvent may range from 0.1 to 60 percent depending the molecular weightof the polymer, the structure of the polymer and the solvent used. Ingeneral, polymer-solvent systems that provide low viscosity (1-500centipoise) or medium viscosity (500-5000 Centipoise) solutions arepreferred. High viscosity solutions, can be used but are difficult toinject and therefore may be less preferred. When using polymer solution,it is understood that polymer-solvent combination chosen may precipitatein vivo in few minutes to several hours depending on the polymer solventcombination chosen. Several factors affect polymer precipitation whichinclude polymer molecular structure, hydrophobicity of polymer, polymermolecular weight, water solubility of polymer solvent chosen, amount ofwater present in tissue site or environment (infected oozing woundstissue sites or bleeding wounds may have significantly more water thannormal skin tissue) and the like). The appropriate polymer-solventcombination must be chosen depending on the desired medical applicationin mind. Factors like biocompatibility of solvents and polymer, how fastthe polymer precipitation is desired, tissue site and the like areconsidered in choosing a proper polymer-solvent combination.Polymer-solvent combination that precipitates in 0.1 to 40 minutes,preferably 0.5 to 30 minutes, and most preferably precipitates in 1 to20 minutes is most preferred. The list of solvents for a given polymercan be accessed from Polymer Handbook or general polymer chemistryliterature or can be determined experimentally. The preferred averagemolecular weight of polymer may range from 1000 to 200000 g/mole, evenmore preferably 5000 to 100000 g/mole. Polymers with molecular weightgreater than 200000 g/mole can be used but their solutions may form highviscosity solutions and their tendency to precipitate quickly can limittheir utility. The list of drugs that can be used is given in definitionsection of this document. The drug may be dissolved, suspended oremulsified before injecting. The concentration of the drug in thepolymer (relative to polymer plus drug weight) may range from 0.1percent to 50 percent, preferably 1 to 40 percent and most preferably 10to 30 percent. The drug may be dissolved or dispersed or emulsified inthe polymer solution. If drug is insoluble in the polymer solventsystem, fine particulates (particle size 0.1 microns to 500 microns) maybe used. The particle size chosen should be less than the needle size ofthe injecting device or artificial cavity size. The polymer may be addeda medical imaging agent or colorant to help the delivery/depositionprocess. The colorant may be dissolved or suspended in the polymersolution, preferably dissolved in the polymer solution. Manybiocompatible colorants can be used and these include but not limitedto: many FD and C dyes or D and C dyes that FDA has permitted to be usedin approved medical devices. Colorants that have been used in absorbablesurgical sutures or contact lens materials are most preferred. Partiallist of coloring agents or coloring compositions is given in thedefinition section of this document. The in vivo biodegradation time forthe polymer may be from few hours to few years, preferably few days to12 months. The deposited particle may release the drug in a sustainedmanner. The delivery of the drug may last for few hours to severalmonths, preferably 3 days to 180 days. The release rate of the drug mayfollow zero order rate release (constant release over a period of time)or may follow standard diffusion model or combination of both. The drugmay be released via diffusion and/or erosion mechanism of the carrier.Various copolymers of polylactones have different in vivo degradationtimes. For example, PLGA (PDLG 5002) is generally suitable for 30-90 daydelivery, PLGA with higher PLA content will have 6 month to one yeardegradation time. Polycaprolactone based polymer generally have 1-2 yearin vivo degradation time. Its copolymer with polyglycolide hasintermediate degradation time depending on the copolymer composition.Some PEG based polylactones have very short degradation times, less than30 days. Those skilled in the biodegradable polymer art will recognizethat many types of biodegradable polymers can be chosen with range ofdegradation time and ultimate choice will depend upon the desiredclinical application. Hydrogels based polymers, especially crosslinkedPEG based hydrogels are generally more suited for protein based drugs.Pluronics, Tetronics and its derivatives gels may be used for short termdelivery upto few hours to few days.

In one illustrative embodiment, a sugar based dissolvable array is usedto create porosity and infuse polymer solution with drug as describedabove to create holes as well as to infuse the solution. Briefly,hyaluronic acid dissolvable array, either purchased from commercialsources or made by casting sodium hyaluronate solution in PBS insilicone rubber mold. The needles of the array are coated with polymersolution (PLGA dissolved in ethyl acetate or polyethylene glycoldimethyl ether molecular weight around 550 g/mole) is coated on theneedles and then inserted into the skin tissue. The polymer solventchosen should be non-solvent for needle material. In this case, NMP is anon-solvent for hyaluronic acid. A combination of polymer solvent andnon-solvent for needle material can be found using a polymer handbook orchemistry handbook. Laboratory solubility tests may be done to make surethat the polymer solvent chosen does not affect/dissolve the microneedlematerial. The water in the tissue dissolve the hyaluronic acid basedneedles and create a space for polymer solution to occupy andprecipitate in situ forming an implant in situ. The precipitated polymerreleases the drug for local and systemic therapeutic effect.Alternatively, the PLGA solution is first applied on the skin tissue anddissolvable microneedle array then pressed on the skin through thesolution. The needles insert the skin tissue and drag the solution withit. After dissolution of needles in the tissue, the dragged solutionoccupies the space created by the needle forming in situ implant forsustained drug delivery. Arrays made from low molecular weight sugars(molecular weight less than 5000 g/mole) are preferred because theyquickly diffuse into tissue and the space created by them can be used asdescribed before.

Injectable Compositions Comprising Neat Liquids

The fluid compositions that can be injected in the cavities may compriseliquid carrier and/or drug. The compositions stay in liquid state in thearray where drug is either suspended or dissolved or combinationsthereof. The fluid carrier used to fill the cavities may be an oil,polymeric or non-polymeric liquid. The liquid carrier is substantiallyliquid at room temperature or around body temperature (37 degree C.).Biocompatible liquid carriers may be hydrophobic or hydrophilic. Theliquid can be oils such as sucrose acetate isobutyrate, vitamin E andits derivatives; fatty acids like oleic acids and its derivatives; fattyalcohols; liquid non-ionic surfactants like polysorbate, Tween® 40 orTween® 80; polymers like liquid polylactones, liquid polyhydroxyacids,liquid PEG-polylactone copolymers, PEO-PPO-polylactone copolymers,liquid polytrimethylene carbonate and its copolymers, liquidpolyorthocarbonates, and its copolymers or combinations thereof and thelike are preferred. Biodegradable liquids are most preferred. The liquidcarriers along with drugs (either dissolved or suspended or emulsified)are delivered in the cavity via injection or other methods describedpreviously. The biodegradable liquids/microparticles used in thisinvention may last in the body from 3 hours to few years, preferablyfrom 24 hours to 360 days, even more preferably from 24 h to 90 days.The drug loading in liquid carriers may range from 0.01 percent to 50percent, most preferably 0.1 percent to 40 percent, even more preferablyfrom 1 to 30 percent. In one illustrative embodiment, sucrose acetateisobutyrate is used a biocompatible liquid carrier and rifampin as amodel drug. The mild color of rifampin is used as a visual aid todeposit the liquid in the cavities.

The liquid deposited in the cavity delivers the drug in a sustainedmanner. In another embodiment, an herbal therapeutic like turmeric isloaded in a vitamin E (loading at 1-10 percent concentration) in thecavities created in the skin tissue.

In another embodiment, non-polymeric liquid sucrose acetate isobutyrateis used as a liquid carrier. In some cases, viscosity-modifying agentssuch as biocompatible organic solvents like ethanol, DMSO and the likemay be added in any proportion (generally 1 to 99 percent, preferably5-90 percent, most preferably around 20 percent) to adjust the viscosityof the non-polymeric liquid carrier like sucrose acetate isobutyrate.The lower or higher viscosity can help the liquid carrier to penetratecavity space created. Other additives such as antioxidants, UVstabilizers, generally found in pharmaceutical preparations may also beadded.

In one embodiment, a liquid biodegradable polymer like polycaprolactoneor PLGA is used as a liquid carrier. Liquid polymeric carriers areespecially useful for sustained delivery of therapeutic drugs. Manyliquid polymeric carriers are known in the art and could be used. Forexample, U.S. Pat. Nos. 5,631,015 and 5,411,554 and references therein,cited herein for reference only, disclose various biodegradable liquidpolymer compositions and methods of their preparation. Such compositionscould be deposited in artificial pores. The viscosity of the liquidpolymers may be adjusted using biocompatible water miscible solventssuch as water or aqueous buffers, dimethyl sulfoxide, n-methylpyrrolidone, ethanol, glycerol, polyethylene glycol, acetone and thelike. Biocompatible polymers, preferably biodegradable polymers may alsobe added to increase the viscosity if needed. The list of preferredbiocompatible solvents is given in earlier section. The solvent could beadded in any proportions; preferably at a concentration of 1-99 percentpreferably 10-90 percent. After deposition in the artificial cavities,the solvent is dispersed by the tissue (if water soluble) leaving behindthe liquid polymer droplet the liquid polymers comprising polyethyleneglycol are most preferred in many applications. One embodiment (example10J) teaches synthesis of PEG polylactone polymer. By changing the molarratio of PEG hydroxy group and cyclic lactone during the synthesis, thedegree of polymerization lactone in the PEG-polylactone polymer ischanged. The molar ratio is adjusted in such a way that the polymerizedproduct is liquid at ambient or body temperature. Some PEO-PPOcopolymers, preferably PEO-PPO-PEO copolymers (Pluronic® or reversePluronic® or Tetronic® polymers from BASF) or their reaction productswith cyclic lactones that are liquid at room temperature could be used.In one illustrative embodiment, a sodium hyaluronate based dissolvablemicroneedle array is used to make cavities. The liquid PEG-PLA (Example10J) or liquid polycaprolactone polymer based compositions are used toinfuse the compositions inside the skin tissue for local drug delivery.

In another illustrative embodiment, 1 g of vitamin E acetate is mixedwith 100 mg of magnesium carbonate stained with tea stain. The darkcolored suspension is used to fill cavities of 10 by 10 array created insheep tissue. The cavities were first made in the tissue and then filledwith the colored injectable compositions based on Vitamin E. FIG. 19Bshows an image of liquid (vitamin E acetate) microimplant array with redcolored liquid microimplants arranged in 10 by 10 array format. Thestained magnesium carbonate is added as a biocompatible biodegradablevisualization agent. The vitamin E used herein is for example only.Other liquid carriers may also be used. Microimplant array size, shape,height, diameter, volume, density and the like can be changed dependingon clinical application as mentioned in earlier sections.

Example 10J shows some illustrative embodiments where liquid carriersare used to fill artificial cavities in tissue or model materials likegelatin with or without drugs. The arrays formed have liquids,preferably hydrophobic liquids used as carriers for drugs. Generallyliquid carriers can help to release drugs for a short duration of time,typically less than 30 days. However, this should not be considered as alimitation of this invention.

Injectable Compositions Comprising Thermoreversible and/or pH SensitiveGels

This invention discloses formation of thermoreversible gels in situwherein the thermoreversible gel microimplants are made inside theartificial tissue cavities. The injectable compositions havingthermoreversible gelation property are used to make microimplant array.The cavities are made first and then thermoreversible compositionscomprising drugs and/or cells are injected in the cavity. The injectedcompositions undergo insitu gelation due to thermoreversible gelationproperty of the composition. The injectable composition may also beloaded inside the injection device capable of injecting the compositionat 10 to 12000 injections per minute if used with oscillating needle.During each injection, the device can deliver 1.0E-02 to 1.0E-16 ml ofinjectable composition. The composition is either heated (below 60degree C.) or cooled (0-20 degree C.) to make it fluid prior toinjection. In one illustrative embodiment, a 33 MP array is used toinject thermoreversible compositions. After injecting the composition,the composition undergoes temperature induced gelation at the injectionsite due to normal body temperature (37 degree C.). The injectablecomposition reservoir of the oscillating needle device can be cooled orheated to make the composition fluid and injectable. The temporaryreservoir may be thermally insulated to keep the injectable compositionin the fluid state. Cold or warm fluid thermoreversible compositionlayer may be first formed on the tissue and while in cold or warm fluidstate, the fluid composition can be then inserted in the artificialcavities as described before. The body temperature (37 degree C.) willgenerally convert such compositions into thermoreversible gel which canrelease the drug in a sustained manner.

In one exemplary embodiment (example 10H), a solution or liquid thatshows thermosensitive gelation behavior may also be used to infuse underthe skin or in the dermis or in the bioprosthesis surface. Thethermosensitive composition is delivered using oscillating needleapparatus or tattoo machine apparatus as described before. Such liquidsmay be preferentially colored prior to the infusion as describedearlier. The thermosensitive liquids normally are fluid during injectionbut undergo gelation as a result of change in temperature. For example,Pluronic F127 copolymer (a PEO-PPO-PEO copolymer with molecular weightof 12000 g/mole) dissolves in cold PBS (below 10 degree C. atconcentration of 20 to 50 percent). At 20 percent or higher (w/v)concentration and at warm temperature (37-45 degree C.), the F-127solution forms a physically crosslinked hydrogel from a cold solution.This process of gelation is called as thermoreversible gelation becausewhen the gel is cooled, it reverts back to Pluronic liquid solution.Pluronic F-127 solution (30 percent W/V in PBS along with eosin Y as reddye for visualization (0.01 percent) along with drug Rifampin (onepercent, w/v) is injected as a cold liquid (0-10 degree C.) using tattoomachine apparatus as described before or using microneedle array such as33 MP array. The Pluronic liquid undergoes thermosensitive gelation atbody temperature and forms a gel, which releases rifampin in acontrolled manner. If necessary, the machine may be modified to keep theneedle and machine cold during injection. The injecting machine may bekept cooled by blowing cool air on the needle to prevent prematuregelation inside the needle or on the tissue. The color of Rifampin andEosin Y serve as coloring agents which helps to see the injected liquidor polymer. In another embodiment, Pluronic F127, chlorhexidine acetatean antibacterial and methylene blue as a coloring agent are dissolved incold PBS wherein Pluronic F127 concentration in the PBS is around 33percent. At this concentration, Pluronic F127 is liquid at 0-15 degreeC. but forms a gel at body temperature. The cold liquid is injected inthe tissue where a change in temperature (0-15 degree C. to 37 degreeC.) causes F127 solution droplets to from gel particles. The gelledparticles deliver the drug compound in a sustained manner. Pluronic F127is generally useful to deliver the compound from few hours to few days.F127 shows thermoreversible gel property at certain concentration range,generally around 15-45 percent w/v concentration range. The gelationtemperature can vary depending on the solutes and drug added, drugconcentration, pH and buffers used and polymer concentration. Artisanscan understand that a formulation must be developed for a given drug andthermosensitive polymer wherein the polymer will show gelation propertyat body temperature upon implantation. It is important that many waterbased compositions described in this invention are osmotically balancedwherein such solution does not create any osmotic imbalance wheninjected inside the body. Some polymers such as some gelatin grades orPEO-polylactone copolymers undergo gelation when injected as a hotsolution (less than 65 degree C., preferably less than 50 degree C.) andcooled as to body temperature (37 degree C.) or at ambient temperaturemay also be used. Many other types of thermosensitive polymers are knownin the art. Among these biodegradable or bio-dissolvable polymers(polymers that dissolve in the human body and removed safely from thebody without harmful effect) are preferred. The thermosensitive polymersthat can be used include but not limited to are: Pluronic or PEO-PPOcopolymers; reverse Pluronics; polyacrylamides such as poly-isopropylacrylamide and their copolymers; gelatin (various grades); chitosanbased compositions and its derivatives, cellulose derivatives, variousPEG-polylactone copolymers, PEG-PLA, PEG-PLHA, PEG-polyhydroxycopolymers, and the like. U.S. Pat. Nos. 6,004,573 and 7,740,877, USpatent application 20140256617 and references therein, cited herein forreference only, disclose thermosensitive gel compositions. Suchcompositions may also be used for deposition inside the artificialcavities. In one illustrative embodiment, a Jeffamine lactide basedthermoreversible composition comprising rifampin encapsulatedmicrospheres is injected in to 4 by 4 array of artificial cavitiescreated in a sheep skin tissue. The compositions form a thermoreversiblegel in the cavity forming microimplant array containing rifampinencapsulated drug (FIG. 22D). The rifampin is released from themicroimplant array in a sustained manner.

In another illustrative embodiment, a Pluronic or PPO-PEO-PPO basedcopolymer (Jeffamine, molecular weight 1900 g/mole) is first reactedwith dl-lactide in presence of stannous octoate to make aJeffamine-polylactide copolymer (Example 10H). The copolymer synthesizedhas thermosensitive gelation properties. A 20-40 percent ofJeffamine-lactide polymer solution in PBS forms gel at 30-40 degree C.and is liquid/fluid around zero degree to 10 degree C. The cold solutionof this polymer along with rifampin loaded microspheres as visualizationagent as well as sustained drug delivery carrier is used for filling theartificial cavities. The cavities are first made in an array form andthe ice cold composition (around zero degree C.) is filled in thecavities using a syringe and needle. At body temperature (37-40 degreeC.), the polymer exists as a solid gel with entrapped microspheres. Themicrospheres release the drug for local or systemic therapeutic effect.FIG. 22D shows the sheep skin tissue with 4 by 4 microarray implantcontaining Jeffamine lactide copolymer thermosensitive gel (an exemplarythermosensitive gel array) and rifampin microspheres entrapped in thegel (red colored 2207).

The thermosensitive compositions described herein can deliver variety ofdrugs including protein drugs. The drug may be microencapsulated in abiodegradable matrix for better control over release profile. Thedetailed list of drugs is given in the definition section of thisdocument. Up to 0.1 percent 30 percent drug may be loaded (relative togel weight) in the thermosensitive composition. Actual loading willdepend upon the type of drug used, drug solubility, type ofthermosensitive polymer used and the like. As stated before, coloring ormedical imaging agent may be added to the thermosensitive composition toassist in the delivery of the composition and to follow its degradationafter implantation. U.S. Pat. No. 7,790,141, cited herein for referenceonly, discloses radio-opaque compositions and such compositions may beadded and used for local delivery as described before.

The illustrative compositions described above are especially useful fordelivery of cells due to physical nature of thermoreversible gelationprocess. The temperature induced transition is generally well toleratedby the cells and therefore can be used for therapeutic use. Osmoticallybalanced solution of thermosensitive polymer in appropriate cell culturemedium or PBS is used to entrap cells in the microimplant array asdiscussed before and entrapped cells in array can be used fortherapeutic use. The microneedle implant array gel compositions withcells as described above may be added with cryopreservative, cast asmicroneedle array and frozen at −80 degree and then implanted in frozenstate in the body to form microimplant array with cells.

In some embodiments, pH sensitive polymer gelation property is used toform a gel in situ inside the cavity. Temperature and pH sensitivepolymers are known in the art (M. Rizwan et al., Polymers, volume 9,page 137, 2017, cited herein for reference only) and such polymers canalso be used to make injectable compositions. Buffered aqueous solutionsof pH sensitive polymers are present as a liquid under mildly acidic (pH4-6.9) or basic aqueous conditions (7.5 to 9) but form gel around pH 7.2or physiological pH. When mildly acidic or basic aqueous solutions areexposed to physiological pH such as pH around 7.4, the polymer in thesolution forms gel and this property can be used in making injectableimplants to form gel based array. Collagen is soluble in mildly acidicsolution but forms a gel when exposed to neutral pH. Copolymers ofn-alkyl acrylamide, particularly n-isopropyl acrylamide with monomerscontaining acidic or basic groups show pH and temperature sensitivegelation. Such systems or polymer systems reported by M. Rizwan et al.may be used in making injectable polymers. Certain blends of chitosanpolymers are also known for pH sensitive gelation and such polymers mayalso be used.

Injectable Compositions Comprising Precursors of CrosslinkableCompositions.

The invention discloses methods and compositions for making microimplantarray in situ inside the live tissue or inside a bioprosthesis tissue.The injectable compositions are precursors of crosslinkable compositionswhich are injected in the artificial cavities to form crosslinkedmicroimplant array. The crosslinked microimplant array may comprise adrug and/or live cells for therapeutic use. In one illustrativeembodiment, precursors that form crosslinked polymer preferablycrosslinked hydrogel structures with or without cells or cellularcomponents or drugs are disclosed. The precursors are formulated asinjectable compositions with or without cells or drugs and then injectedin the tissue using oscillating needle apparatus or hollow microneedlearray such as 33 MP.

The precursors react with themselves or components in the tissue and/orwith external stimulus such as light that trigger a chemical reaction orcrosslinking reaction forming crosslinked polymers in the artificialcavities. The crosslinking reaction converts the injected compositionsinto solids or hydrogels entrapping cells and/or drugs. The encapsulatedcells or drug provide therapeutic benefit. Preferably the crosslinkedstructures are biodegradable.

In one illustrative embodiment (example 10D), a biodegradablemacromonomer is synthesized and then formulated to make an injectablecomposition which can be initiated by long UV light or visible light. Apolyethylene glycol based water soluble biodegradable macromonomer(precursor) is prepared by initiating a cyclic lactone polymerizationfrom the hydroxyl groups of PEG starting material. The PEG lactatepolymer is then endcapped by with polymerizable acrylate group. This isachieved by reacting the PEG-lactate diol with acryloyl chloride usingtriethyl amine as a base catalyst. The PEG-lactate-acrylate is designedto be water soluble (PEG to lactide weight ratio is kept high tomaintain water solubility) and can undergo polymerization at 10 percentor higher concentration (above its critical micelle concentration inwater) in water or water based buffers such as PBS buffer (pH 7.4). ThePEG-lactate-acrylate solution is mixed with photoinitaitor solution(either UV light photoinitaitor or visible light photoinitaitor). Theprecursor solution along with photoinitiator is sterile filtered anddeposited using a tattoo machine or other oscillating needle apparatusor using hollow needle array or using standard syringe and fine needle.The machine deposits small droplets of mixture in the tissue (dermistissue) if used with oscillating needle. The composition can also beinfused in artificial cavities created using methods as describedbefore. The polymerization of composition in the cavities is triggeredby illuminating the composition with long UV light or with visible light(514 nm). The compositions can be irradiated with light duringdeposition process as long as liquid compositions in the device areprotected from light. The illustrative composition undergoespolymerization and crosslinking triggered by light and photoinitaitor in5 to 400 seconds. The polymerization reaction converts the liquidcomposition into solid crosslinked biodegradable hydrogel particlesentrapping the drug or cells in the crosslinked hydrogel. Thecrosslinked hydrogel degrades in 2-9 months due to hydrolysis of lactategroup. One advantage of photopolymerization systems is that the systemcan be used to deliver live cells for therapeutic use without damagingthem. The cells could be therapeutic cells or stem cells or any othercells as described in the definition section. The cells also could beused for tissue engineering application. The degraded hydrogel fragmentsare safely removed by the body. U.S. Pat. Nos. 5,529,914 and 5,410,016,cited herein for reference only, can provide additional compositions andmethods for photopolymerizable, biodegradable or biostable hydrogels andtheir use in cell encapsulation. Many polymerizable precursors are knownin the prior art and can be deposited and crosslinked using the methoddescribed in this invention. Protein based macromonomers such ascollagen, keratin or albumin can be modified with photopolymerizablegroups and crosslinked in situ using methods described in thisinvention. In another exemplary embodiment, 200 mg of PEG35K-lactate-acrylate macromonomer prepared according to procedure shownin Example 10D is dissolved in 800 mg PBS. After complete dissolution,200 mg of magnesium carbonate is added as opacity creation agent or as avisualization agent. 300 mg Irgacure 2959 is dissolved in 700 mgn-methyl pyrrolidone. 5 microliters of Irgacure 2959 solution is addedto the macromonomer solution. The sterile solution (precursor solution)is then filled in the array of cavities (4 by 4 array, manually createdusing 24 gauge needle) using syringe needle in the sheep tissue, excesssolution is wiped off and exposed to long UV ultraviolet light(Black-Ray UV lamp, 360 nm light, 10000 mW/cm2 intensity) for 5 minutesto photopolymerize and crosslink the macromonomer solution to form acrosslinked hydrogel. Crosslinked biodegradable hydrogels 4 by 4microimplant array in sheep tissue is shown in FIG. 19A. The crosslinkedhydrogel 4 by 4 hydrogel array with magnesium carbonate as visualizationagent/filler (2401) is clearly seen in the image. In another embodimentas above, magnesium carbonate is replaced with fibroblast cell pelletwith 1000000 human foreskin fibroblasts cells and the mixture is filledin the artificial cavities of the array and exposed to UV light to formcrosslinked gel with live cells.

Another embodiment (Example 10E) describes condensation polymerizationof precursors, preferably PEG based precursors. In this illustrativeembodiment, NHS ester of PEG and albumin or trilysine are mixed to forma precursor solution. The mixed solution is then deposited inside thetissue cavities using microneedle array or tattoo machine like device oroscillating needle apparatus. The deposition is done prior to completecrosslinking or change in viscosity or gelling the solution. Prematurecrosslinking can prevent the deposition and is generally avoided. It ispreferred that the composition is mixed just prior to infusion and usedimmediately. In the preferred embodiment, the precursor solutions aremixed inside the oscillating needle apparatus in a mixing chamber andused immediately for the infusion inside the tissue cavities. In oneillustrative embodiment (Example 10E), PEG NHS ester and albuminsolutions are mixed in PBS (pH 7.2) and used. The composition that formsgel in 30-60 seconds and is injected using this apparatus beforegelation. A small amount of triethanol amine may be added to acceleratethe gelation process. Many types of condensation polymerization systemsare known in the art and such reactions can be used making gel particlesin situ as described in this invention. U.S. Pat. Nos. 6,887,974,7,592,418, and 6,323,278 and cited references therein, cited here forreference only, can provide various compositions that can be polymerizedin situ using condensation polymerization method. Other precursors thatcan be used for in situ polymerization used include but not limited to:precursors that form crosslinking by the reaction of isocyanate andalcohols or amine and epoxide or acrylate and amine, acrylate and thioland the like may also be used. In general, precursors have nucleophilicand electrophilic reactive groups and the total number of reactivegroups in the precursors must be greater than or equal to five. Ioniccrosslinking such as crosslinking of sodium alginate solution (0.2percent solution in deioinzed water) with calcium chloride solution (2percent in distilled water) can also be used. In this case, a two-needledelivery system is used or multilumen needle is used. One needle orlumen delivers the 1 percent sodium alginate solution and anotherneedle/lumen delivers calcium chloride solution. The interaction of twodroplets triggers ionic crosslinking of sodium alginate formingcrosslinked calcium hydrogel particle in situ.

In another embodiment (Example 10F), the precursors react via enzymaticpathway to form a crosslinked (physically and chemically crosslinked)compositions. Fibrin glue based microimplant arrays are used as anillustration of enzymatically formed microimplant array. In thisillustrative embodiment fibrin glue microimplant arrays are formed insitu. Briefly fibrin glue precursors are deposited prior to gelation inthe tissue cavities or using tattoo machine inside the tissue. Theprecursors compositions react with each other forming fibrin gluehydrogel microimplants in situ inside the artificial cavities in tissue.Fibrin glue formation is a complex enzymatic reaction. The solution ofconcentrated fibrinogen and factor XIII are combined with a solution ofthrombin and calcium. Once the thrombin/calcium is combined with thefibrinogen/factor XIII, a fibrin clot forms in few seconds to fewminutes, depending on the thrombin concentration, temperature, calciumion concentration, fibrinogen concentration and the like. The fibringlue components are mixed and deposited inside the tissue cavity priorto gel or clot formation (within few seconds). The factor XIII in theformulation continues to act for several days leading to covalentlycrosslinked fibrin gel. If drugs are entrapped in the fibrin clot, thoseare then released from the fibrin clot via diffusion and/orbiodegradation process. In the preferred formulation, the fibrin glue iscolored for improved visualization. Alternatively, precursors of fibringlue can be delivered using multilumen needle or bi-needle basedoscillating machine similar to described for alginate gel making. Fibringlue and PEG based biodegradable hydrogels described above areespecially useful for delivery of protein drugs like growth factors ortherapeutic cells. U.S. Pat. No. 8,557,535 and references andcross-references therein; describe some fibrin glue compositions, citedherein for reference only. Such compositions could also be used forlocal delivery of fibrin glue based compositions described above. Theprecursor solutions may be preferably deposited using a multilumenneedle as described before. For example, solution comprising fibrinogenmay be fed via one lumen and the solution comprising thrombin may be fedby another lumen. Both the solutions may exit at the same time, mixed insitu and react to form a crosslinked material in situ. Fibrin glue maybe especially suitable for delivery of cells. The therapeutic cells suchas stem cells may be mixed with fibrinogen solution and the solution iscrosslinked by reacting with thrombin as described above. The entrappedcells in the crosslinked network may provide therapeutic effect. Thecrosslinkable precursor compositions described as above may also bedeposited using hollow microneedle array such as 33 MP array asdescribed previously. The compositions are delivered

The amount of drug that can be injected may range from 0.1 percent to 30percent, preferably 1 to 10 percent depending on the drug to bedelivered and disease that has been addressed. The size of hydrogelparticles will depend on the artificial cavity size.

Injectable Compositions Comprising Cells

In some embodiments, the technology described herein can be implementedby using injectable compositions that comprises live cells, preferablylive mammalian cells, or cellular elements thereof. Cellular elements,which can be used for therapeutic use, include, but are not limited tomammalian cells including stem cells; cellular components or fragments,enzymes, DNA, RNA, and genes may also be included as bioactivecomponents or drugs. A method for local delivery of an injectablecomposition can include obtaining precursors that form crosslinkedcompositions in situ wherein the volume of crosslinked compositionformed is less than 1.0E-02 ml. The crosslinked compositions maycomprise of cells, drugs, or imaging agents. The injectablecomposition(s) are loaded inside the injection device capable ofinjecting the composition at 10 to 12000 injections per minute. Duringeach injection the device can deliver 1.0E-02 to 1.0E-16 ml ofinjectable composition. After injecting the composition, the injectedprecursors undergo ionic, physical, chemical or enzymatic reaction suchas polymerization, ionic or covalent crosslinking, and thermoreversiblegelation and the like forming a physically, ionically or chemically orenzymatically crosslinked material and entrapping the cells withoutsubstantially affecting their viability. The crosslinked material couldbe hydrophobic or hydrophilic or hydrogel. The crosslinked materialformed as above could be biostable or biodegradable.

The invention discloses methods and compositions for making encapsulatedmicrospheres/microspheres in situ inside the tissue or inside abioprosthesis tissue. In one embodiment, precursors that formcrosslinked polymer preferably crosslinked hydrogel structures with orwithout cells or cellular components or drugs are disclosed. Theprecursors are formulated as injectable compositions with or withoutcells or drugs are injected in the tissue using oscillating needleapparatus as small droplets. The precursors react with themselves orcomponents in the tissue or with external stimulus such as light thattrigger a chemical reaction or crosslinking reaction forming acrosslinked structures. The crosslinking reaction converts the injecteddroplets into solids or gels entrapping cells or drugs. The encapsulatedcells or drug provide therapeutic benefit. Preferably the crosslinkedstructures are biodegradable. The crosslinked structure could behydrophobic or hydrogels or hydrophilic.

One advantage of photopolymerization systems is that the system can beused to deliver live cells for therapeutic use. The cells could betherapeutic cells or stem cells or any other cells. The cells also couldbe used for tissue engineering application. The degraded hydrogels aresafely removed by the body. U.S. Pat. Nos. 5,529,914 and 5,410,016,cited herein for reference only, can provide additional compositions andmethods for photopolymerizable biodegradable or biostable hydrogels andtheir use in cell encapsulation. Many polymerizable precursors are knownin the prior art and can be deposited and crosslinked using the methoddescribed herein. Protein based macromonomers such as collagen, keratinor albumin can be modified with photopolymerizable groups andcrosslinked in situ using methods described in this invention.

Fibrin glue and PEG based biodegradable hydrogels described above areespecially useful for delivery of protein drugs like growth factors ortherapeutic cells. U.S. Pat. No. 8,557,535 and references andcross-references therein; describe some fibrin glue compositions, citedherein for reference only. Such compositions could also be used forlocal deliver of fibrin glue based compositions described above. Theprecursor solutions may be preferably deposited using a multilumenneedle as described before. For example, the solution comprisingfibrinogen may be fed via one lumen and the solution comprising thrombinmay be fed by another lumen. Both the solutions may exit at the sametime, mixed in situ and react to form a crosslinked material in situ.Fibrin glue may be especially suitable for delivery of cells. Thetherapeutic cells such as stem cells may be mixed with a fibrinogensolution, and the solution is crosslinked by reacting with thrombin asdescribed above. The entrapped cells in the crosslinked network mayprovide therapeutic effect.

In some embodiments, a method of forming an implant in a tissue caninclude: providing an injectable composition including live mammaliancells suspended in an aqueous solution; and injecting the injectablecomposition into the tissue at the rate of about 10-12000 injections perminute. In some aspects, the aqueous medium is a phosphate bufferedsolution or Minimum Essential Medium. In some aspects, the aqueousmedium is osmotically balanced. In some aspects, the aqueous compositioncomprises a visualization agent. In some aspects, the visualizationagent is a colored compound, a fluorescent compound, an x-ray imagingagent, or a MRI agent. In some aspects, the colored compound is dye orpigment/microparticle that is biocompatible. In some aspects, thecolored compound is water soluble preferably at physiological pH (PHaround 7.2). In some aspects, the colored compound is selected from thegroup comprising methylene blue; Eosin Y; fluorescein sodium; ferricammonium citrate; D&C Blue No. 9; D&C Green No. 5; FD&C Blue No. 2; D&CBlue No. 6; D&C Green No. 6; D&C Red No. 17; D&C Violet No. 2; D&CYellow No. 10; indocyanine green; rose bengal; phenol red andphenolphthalein. In some embodiments, the derivatives of biocompatiblecolored compounds as above with biocompatible polymeric materials likedextran, hyaluronic acid, albumin or polyethylene glycol and the likemay be used as colored or fluorescent compound. Such derivatives may bemade by using complexation, covalent bonding or electrostaticinteractions with the polymeric materials. In some respects, coloredbiodegradable microparticles described in this invention may also beused as coloring composition.

In some aspects, the method includes injecting the injectablecomposition by a microneedle. In some aspects, each injection of theinjectable composition per microneedle includes about 1 to about 10million live mammalian cells. In some aspects, each injection of theinjectable composition per microneedle includes about 1 to about 10,000live mammalian cells. In some aspects, the live mammalian cells have aviability from about 30% to about 100%. In some aspects, the livemammalian cells have a viability of live mammalian cells from about 35%to about 99.5%. In some aspects, the live mammalian cells have aviability of live mammalian cells from about 40% to about 99%. In someaspects, the cell comprising injectable composition is injected in atissue that is a live tissue or a bioprosthetic tissue. In some aspects,the live tissue includes: adrenal gland tissue, duct cell tissue,sensory transducer cell tissue, placental tissue, iris tissue,cancellous bone tissue, pia-arachnoid tissue, cardiac valve tissue,pituitary gland tissue, fibrocartilage tissue, spleen tissue, bonemarrow tissue, compact bone tissue, peritoneal tissue, liver tissue,retinal tissue, cardiac muscle tissue, tendon tissue, pericardialtissue, pain sensitive tissue, gastrointestinal gland tissue, ectodermaltissue, squamous tissue, neuronal tissue, pleural tissue, lymph glandtissue, ependymal tissue, mesodermal tissue, endodermal tissue, germcell tissue, thyroid gland tissue, lymphatic duct tissue, synovialtissue, epididymis tissue, intervertebral disc tissue, blood celltissue, sclera tissue, gall bladder tissue, renal tissue, cochleartissue, dental tissue, hyaline cartilage tissue, adipose tissue, thymustissue, blood vessel tissue, serosal tissue, autonomic neuron tissue,peripheral nervous system tissue, optic tissue, ocular lens tissue, stemcell tissue, pulmonary tissue, vas deferens tissue, testicular tissue,respiratory gland tissue, smooth muscle tissue, dural tissue, fetalmembrane tissue, umbilical tissue, cranial nerve tissue, ligamenttissue, choroid plexus tissue, autologous tissue, parathyroid glandtissue, ciliary tissue, ovarian tissue, elastic cartilage tissue,skeletal muscle tissue, glial tissue, heart tissue, and combinationthereof. In some aspects, the live mammalian cells are human foreskinfibroblasts. In some aspects, the human foreskin fibroblasts areincluded in a carrier matrix as the injectable composition. In someaspects, the carrier matrix includes a fibrin sealant, a water solublepolymer or monomer thereof or macromonomer thereof, or a thermosensitivegel. This invention discloses several illustrative embodiments whereinlive cells are entrapped/encapsulated in the artificial cavities createdin the live tissue. Several exemplary embodiments in Example 15 disclosepreferred methods and compositions comprising live cells in theartificial cavities. The type of cells and other variables used in theExample 15 is for illustration only and does not limit the invention tospecific embodiments. Example 15 discloses use of illustrative cells fortherapeutic use. Mammalian cells like human foreskin fibroblasts (HFF)are isolated and grown using standard mammalian tissue culturetechniques known in the mammalian/human cell culture art. The cells aretypically grown on tissue cultured flasks which have special surfacetreatments that enable these cells to grow on the flask surfaces. Thetechniques for growing and culturing human cells is well known inmammalian tissue culture/engineering prior art. The HFF cells areisolated and suspended in an exemplary carrier matrix like fibrinsealant or synthetic materials like PEG based macromonomers orthermosensitive gels. The cell suspension is mixed with precursor offibrin glue or PEG based macromonomer precursors. The suspension is theninjected in the live tissue or bioprosthesis tissue like sheep dermaltissue using hollow microneedle array. A 3 by 3 array (33 MP) is used asan example. This array has 9 microneedles and common reservoir for allthe needles to access to and inject. The cell suspension is filled inthe syringe and attached to the array hub via its female Luer lock. Thearray needles are inserted inside the live or bioprosthesis tissue wherethey create cavity first and cells are injected in the cavities createdby the array. The size of the cavity created is same as the size of theneedle and depth of penetration is the height. The precursor of fibringlue undergoes physical/chemical change (crosslinking reaction) to forma fibrin clot (a reaction product of fibrinogen, thrombin, Factor 8,calcium and other materials present in the precursor composition). Theentrapped cells injected in vivo can survive the cavity fillingoperation and can form a microimplant array in the tissue with livecells. The live tissue provides necessary nutrients for cell to functionand produce a therapeutic effect. In some cases, carrier matrix useddoes not provide therapeutic effect, but is added to provide mechanicalintegrity and volume to the cells. Cell suspension containing 40-100percent, preferably 80-100 percent viable live cells in biocompatiblemedium like PBS, MEM and the like may also be used with or withoutcarrier matrix like fibrin glue or PEG based crosslinked matrix. Eachinjection of cell suspension per microneedle may comprise 1 to 10million live cells, preferably 1 to 1 million cells, even morepreferably 1-10000 cells in a suitable medium such as PBS or cellculture medium. The viability of cells used may range from 30 to 100percent, preferably 35 to 99.5 percent and even more preferably 40 to 99percent. The HFF cells may proliferate and form a collagen rich tissuewhich may be helpful in application like healing burn wounds or othertype of wounds or may be useful in cosmetic application. HFF based cellsare currently grown outside in the lab and cells and its extracellularmatrix is used as burn dressing. This process is expensive and requiresseveral days of culturing and specialized sterile handling. In thisinvention, cells are cultured inside the tissue cavity for therapeuticeffect, thereby eliminating the culturing and growing of cells in thelaboratory and its sterilization and packaging costs for the consumer.

Live cells may be injected in the bioprosthetic or live tissue whichinclude but not limited to: adrenal gland tissue, duct cell tissue,sensory transducer cell tissue, placental tissue, iris tissue,cancellous bone tissue, pia-arachnoid tissue, cardiac valve tissue,pituitary gland tissue, fibrocartilage tissue, spleen tissue, bonemarrow tissue, compact bone tissue, peritoneal tissue, liver tissue,retinal tissue, cardiac muscle tissue, tendon tissue, pericardialtissue, pain sensitive tissue, gastrointestinal gland tissue, ectodermaltissue, squamous tissue, neuronal tissue, pleural tissue, lymph glandtissue, ependymal tissue, mesodermal tissue, endodermal tissue, germcell tissue, thyroid gland tissue, lymphatic duct tissue, synovialtissue, epididymis tissue, intervertebral disc tissue, blood celltissue, sclera tissue, gall bladder tissue, renal tissue, cochleartissue, dental tissue, hyaline cartilage tissue, adipose tissue, thymustissue, blood vessel tissue, serosal tissue, autonomic neuron tissue,peripheral nervous system tissue, optic tissue, ocular lens tissue, stemcell tissue, pulmonary tissue, vas deferens tissue, testicular tissue,respiratory gland tissue, smooth muscle tissue, dural tissue, fetalmembrane tissue, umbilical tissue, cranial nerve tissue, ligamenttissue, choroid plexus tissue, autologous tissue, parathyroid glandtissue, ciliary tissue, ovarian tissue, elastic cartilage tissue,skeletal muscle tissue, glial tissue, heart tissue and combinationthereof.

In another illustrative embodiment, a PEG based macromonomer is used asa precursor and as a synthetic hydrogel carrier to encapsulate cells inthe artificial cavities created by the array. PEG based macromonomersthat are biodegradable and used for cell encapsulation have beenreported in U.S. Pat. Nos. 5,801,033 and 5,626,863 and referencestherein, cited herein for reference only. Compositions and methodsreported in U.S. Pat. Nos. 5,801,033 and 5,626,863 may be used toencapsulate cells and inject in the artificial cavities. In oneillustrative embodiment (Example 15), the macromonomer solution withvisible light initiator and co-catalysts and comonomers along with cellsare injected in the artificial cavities created by the array. Thecavities may be partially or completely filled with the injectablecomposition with cells. The cavity volume may be filled 5 to 100percent, preferably 10 to 95 percent even more preferably 70-95 percentwith injectable composition. The macromonomer liquid composition isexposed to green laser light to initiate polymerization and crosslinkingreaction which form crosslinked degradable gels and entraps the cells.The polymerization and macromonomers do not significantly affect theviability of cells before and after encapsulation. The size/shape of theimplant is generally same as the size/shape of cavity of microneedle ofthe array. It is preferred that the hydrogel composition used will notswell (hydrogel absorbs water from the surrounding tissue) excessivelyafter crosslinking reaction or gel formation. It is understood that bychanging variables like the array needle size, number of needles, needleinternal diameter, needle length, number of injections made and thelike, variety of microimplant array size can be created inside the liveor bioprosthesis tissue. Stem cells which can be converted into any typeof cells provided proper chemical and biological stimulus is given. Stemcells are most preferred for therapeutic use. Hollow array like deviceused in illustrative embodiments is for example only and is not alimitation. Other devices and methods described in this invention, knownin the art or yet to be discovered may also be used. Dissolvable orbiodegradable polymer based microneedle arrays, hollow microneedlearray, laser based cavity creation methods are preferred methods forcell based therapies.

The use of fibrin glue, gelatin and PEG macromers for cell encapsulationin the artificial cavities is for illustration only. Other methods knownin the cell encapsulation art such as sodium alginate and calcium ioncrosslinking chemistry, chitosan, protein or peptide based gelationsystems and the like known in the art or yet to be discovered may alsobe used as long as such methods are able to infuse the cells in cavitieswithout affecting their viability and encapsulation matrix isbiocompatible and/or biodegradable.

Some embodiments disclose methods and compositions for preparation ofmammalian cell containing dissolvable array. In one illustrativeembodiment, a mammalian cell suspension (suspended in tissue culturemedium or PBS containing 10 percent dimethyl sulfoxide as acryopreservative agent) is poured into silicone rubber mold (MPatchMicroneedle array mold as an example), which has cavities that cancreate microneedle arrays. The suspension may be centrifuged to fill thecavities completely. The mold with liquid suspension is then frozenbelow the melting point of liquid (PBS or saline solution) to formfrozen solid matrix without forming ice crystals without significantlyaffecting cell viability. The cells can survive freezing process forshort period of time. The frozen cell containing array is removed fromthe mold and is then inserted in the frozen condition in the skintissue. The body temperature dissolves the water in the array needle andcells are released inside the dermal or epidermis or other tissuelayers. In some embodiments, non-toxic biocompatible additives suchpolyethylene glycol, hyaluronic acid sodium salt, carboxy methylcellulose and other materials used in dissolvable microarray can beused. Such additives help to improve mechanical properties of frozensolids without affecting cell viability. Including all additives andinjectable composition materials, the cell suspension should begenerally osmotically balanced to maintain cell viability. Majority ofthe culture medium contain water which can be hard when frozen. Thesharp needle shape and its hardness enable the frozen microneedles topenetrate the tissue surface and deliver the cellular cargo upon meltinginside the tissue.

Mammalian cells can be preserved by freezing (also generally referred ascryopreservation) and reused by thawing. Generally, mammalian cells arebest preserved at −80 degree C. or lower, and at −50 to −70 degree forshorter period of time. Preserved cells are usually stored in liquidnitrogen or around that temperature. The freezing operations must bedone carefully and conditions may vary for type of cells used. A use ofcryopreservation agent such as dimethyl sulfoxide is generallyconsidered as essential when cells are subjected to cryopreservation. Itis generally added at 5-10 percent concentration in the PBS or culturemedia without no magnesium, calcium, or phenol red. The amount of agentadded will depend on the type of cryopreservation agent used. Thecryopreservation agent is believed to prevent ice crystallization in thelive cell structure which can lead to cell death and affect viability offrozen cells. Many cryopreservation agents can be used which include butnot limited to: dimethyl sulfoxide, glycerol, polyvinyl pyrrolidinone,polyvinyl alcohol and the like. Among these, dimethyl sulfoxide is mostpreferred. Commercial cryopreservation medium such as Recovery™ CellCulture Freezing Medium or Synth-a-Freeze® Cryopreservation Medium fromGibco or other vendors may also be used.

It is preferred that cells entrapment is done at the time of therapy orduring a surgical procedure. Specialized sterile kits that can handlecells and injectable compositions may be designed and supplied to beused during a surgical procedure.

In some embodiments, thermoreversible compositions as describedpreviously may be used to encapsulate cells. Thermoreversiblecompositions based on PEG-polylactones, Jeffamine-lactide, gelatin,chitosan based compositions, and poly-n-alkyl acrylamide orpoly-n-isopropyl acrylamide may be used for cell delivery inside thecavity.

In one embodiment, a PEG based macromonomer (Example 20B or Example 21)is dissolved in Synth-a-Freeze® Cryopreservation Medium from Gibco orPBS containing 10 percent DMSO. All operations are carried out insterile condition and all solutions are sterilized prior to use. Eosin,vinyl pyrrolidinone and triethanoll amine are added as visible lightinitiator and cocatalyst. The macromonomer solution is cooled inrefrigerator (4-10 degree C.). Live cell suspension is first centrifugedfor 100-200×g for 5 to 10 minutes, supeinatant medium is removed almostcompletely leaving behind mostly cell pellet. The cell pellet isre-suspended in cold precursor solution as above and filled in the coldsilicone based microneedle array mold (MPatch Microneedle array) whichis precooled to 4 degree C.) and exposed to visible light for 30-120seconds to polymerize and crosslink the precursor to form a crosslinkedhydrogel with encapsulated cells. The array with cells is frozen to −80degree C. at the rate of one degree per minute. At the time of use, thearray is removed in frozen state, thawed to −10 to zero degree C. (usinga polyester adhesive backing tape) and pressed in the skin tissue andthe backing tape is removed leaving the array inside the tissue. Thefrozen state provides shelf life for the cells as well as hardness tothe hydrogel matrix which is sufficient to penetrate the tissue. Thecryopreservation agent helps to maintain cell viability in frozen state.The crosslinked hydrogel provides Immunoprotection (the crosslinkednetwork prevents diffusion of immunoglobulins (molecular weight rangearound 150000 Daltons) to the cells but allows diffusion of smallmolecular weight nutrients and cellular waste products. Mammalian cellcontaining arrays can be used for variety of therapeutic use.

Microimplants comprising live cells may be formed first and thenimplanted using AIA device as discussed before. Briefly cell andinjectable cell encapsulation matrix are mixed together and filledinside the mold of suitable size and shape wherein the mold size issmaller than AIA device cavity. The encapsulation matrix forms a gelwithout substantially affecting the viability of cells. The formedimplants with live cells are then loaded in the AIA device anddeployed/implanted in the tissue as discussed in this invention (pushedusing plunger array). Alternatively, cells may be microencapsulated inthe microspheres (size generally less than 500 microns, preferably lessthan 300 microns). The encapsulated cells are then injected in theartificial cavities or in porous microimplants and then implanted. Livecells may also be grown on porous microimplants such as EDC crosslinkedcollagen or gelatin, fibrin glue and the like and such implants may bedeployed and implanted using AIA device as discussed in this invention.

Microneedle Array Comprising Crosslinked Biodegradable Hydrogels orPolymers

Biodegradable hydrogels have found several medical applications. In themicroneedle array format, the hydrogels can be more easily deliveredunder the skin. However, most hydrogels have poor mechanical properties.Hydrogel materials commonly used in the art are not degradable butdissolve away after implantation. The synthetic polymers used in theprior art such as polyvinyl pyrrolidinone, must have low molecularweight. High molecular weight polyvinyl pyrrolidinone (molecular weightgreater than 100000), polyethylene glycol (molecular weight greater than35000) cannot be eliminated from the body and therefore cannot be usedin array preparation where substantial biodegradation is necessary.Generally, strength of array depends on its molecular weight. In thisinvention, methods and compositions are provided wherein highermolecular weight synthetic polymers such as polyethylene glycol can beused in making array materials. The implantable array materials are madeusing very high molecular weight crosslinked materials and arebiodegradable in nature. Such array materials, when fabricated andimplanted under the skin, undergo biodegradation and/or hydrolysis whichconverts crosslinked materials into small molecular weight fragmentswhich then can be eliminated from the human or animal body.

In one illustrative embodiment, Example 20, a biodegradable macromonomermade using 10000 molecular weight polyethylene glycol linked topolylactide which is linked to polymerizable acrylate group at both theterminal ends. The solution of this macromonomer is added in molds ofmicroarray cavities. The macromonomer in the solution is polymerized viaacrylate group increasing its molecular weight several times, typicallygreater than 2-100 times. The crosslinked gel is dried, removed from themold. The array has better mechanical properties than its monomercounterpart due to increased molecular weight via crosslinking reaction.The array is implanted in the skin tissue and the polylactate undergoeshydrolysis upon implantation which reduces the molecular weight of thecrosslinked polymer to its monomer fragment which then can be eliminatedfrom the tissue. The crosslinked polymer hydrogel also can entrapvariety of drugs, especially protein based drugs which could be releasedin a sustained manner upon implantation. U.S. Pat. No. 6,306,922, citedherein for reference only, discloses additional macromonomer basedcompositions which produce crosslinked hydrogels that could becrosslinked and used to make implantable microneedle array. By changingthe molecular weight of macromonomers polyethylene glycol orbiodegradable polymer unit; its biodegradable polymer type (polylactideis changed to polycaprolactone or to polyglycolate or topolytrimethylene carbonate or combinations thereof) and number ofpolymerizable groups per macromonomer, crosslinked hydrogels withvarious degradation time and molecular permeability can be synthesized.

In another embodiment, crosslinked polyethylene glycol based crosslinkedhydrogels made using condensation polymerization method is used. PEGderivative with degradable glutarate group and terminal reactive group(n-hydroxysuccinimide, NHS, exemplary electrophilic group) is reactedwith equimolar quantities with PEG derivative with terminal amine groups(exemplary nucleophilic group). The polymerization and crosslinkingreaction is carried out under equimolar concentration of reactive groupsin water under close to physiological conditions (pH around 7.4, totalreactive groups greater than or equal to 5 for crosslinking to occur) insilicone mold cavities for array preparation. PEG amine and NHS groupsreact forming amide bonds and increase the molecular weight viacondensation polymerization and crosslink to form a gel. The gel isdried, removed from the mold and array is inserted in the body. Uponimplantation, the glutarate ester bond in the crosslinked hydrogelundergoes hydrolysis in the body reducing the molecular weight ofcrosslinked hydrogel. The hydrolyzed fragments are removed from thebody. Additional examples of crosslinked degradable materials can befound in U.S. Pat. Nos. 7,009,034 and 6,534,591, cited herein forreference only. The crosslinked compositions with wide range ofdegradation profile from few days to few years can be made by properchoice and number of nucleophilic and electrophilic group, PEG molecularweight, reaction conditions (time, temperature, buffers etc.) and use ofdifferent degradable esters like succinate, glutarate, adipate, suberateor their combinations and the like. The polymerized/crosslinked gels asdescribed above can be made with various degradation profiles suited forvariety of drug delivery applications.

In some embodiments, arrays were made from natural polymers likecollagen, gelatin. These polymers could be used as crosslinked ornon-crosslinked materials and their array upon implantation degrades viaenzymatic degradation pathway.

The crosslinked polymer disclosed could also be hydrophobic andbiodegradable. The hydrophobic structures include crosslinkable polymerssuch as hydrophobic macromonomers made by polymers or copolymers ofpolylactones or polyhydroxyacids and polycarbonates. Such polymers aremay be oligomers of polylactones which are endcapped with polymerizablegroups such as acrylate or methacrylate group and generally present asneat liquids. These neat liquid oligomers crosslink via polymerizablegroups producing crosslinked hydrophobic polylactone based crosslinkednetwork. Additional examples of hydrophobic liquid oligomers that can bepolymerized by free radical polymerization can be found in U.S. Pat. No.6,352,667, cited herein for reference only. The liquid precursors ofsuch polymers are poured into silicone mold cavities as discussed beforeand then crosslinked. Prior to precursor crosslinking a formulationcompatible visualization agent may be added to aid array implantation.The crosslinking of hydrophobic precursors produces hard sharp edgedmicroneedle biodegradable array which can be used for therapeutic drugdelivery.

This invention is not limited to application on skin tissue. Minimallyinvasive surgical devices (MIS) based methods can also be used to createporosity at a local site accessed using MIS and then accessed site canbe treated using compositions and methods described in this invention.For example, porosity may be created using angioplasty balloons attachedwith flexible microneedles. Injectable compositions then can be appliedon the surface and then infused using the needles on angioplastyballoons. Laparoscopy based methods may be used to treat areas inabdominal cavity. It is understood that a MIS device modifications maybe made for a given disease that is managed and such modifications areconsidered as part of this invention.

Injectable Compositions Comprising Biocompatible and BiodegradableInorganic and Polymeric Fillers:

Polymer Solutions Based and Thermoreversible Gelling CompositionsComprising Fillers:

The biodegradable polymer solution in water miscible organic solvent canbe used for sustained drug delivery. Such compositions can be deliveredusing syringe and injected via intramuscular injection. The injectedpolymer undergoes precipitation forming implant in situ afterdissipation of water soluble solvent in the tissue. In this invention,the injectable composition comprising polymer solution is improved byaddition of biodegradable and biocompatible filler particles in theinjectable composition. FIG. 21 shows schematic of a method for in situimplant formation in the human or animal body comprising biodegradablefillers. 2101 schematically represents an injectable compositioncomprising a drug and biodegradable polymer in water miscible organicsolvent or crosslinkable precursor composition/s comprising a drug or athermoreversible polymer composition in aqueous solution or polymermelt. 2102 comprises a biodegradable, biocompatible inorganic or organicfiller microparticles that are insoluble in the injectable composition2101. The components of 2101 and 2102 are mixed to form asuspension/emulsion and injected into human or animal body viaconventional syringe or using methods described in this invention toform implantable arrays. The injected composition undergoes physicaland/or chemical change (precipitation, crosslinking, cooling,thermoreversible gel formation and the like) entrapping the drug and thefiller in the formed implant. The presence of filler is believed toprovide nucleating sites for polymer precipitation as well as providemore surface area for the implant formation/precipitation therebyaltering drug release profile. Filler also change mechanical propertiesof the precipitated polymer which helps to push out from “array inarray” apparatus described in this invention. The filler can also affectlocalized pH changes depending on the type of filler used. For example,magnesium carbonate provides local basic environment.

FIG. 21A shows steps involved in making the implant with the filler. Inone embodiment, magnesium carbonate (particle size less than 300microns) is used as an illustrative filler. PLGA polymer (PDLG 5002) isdissolved in DMSO along with methylene blue as a colorant. The polymersolution is mixed with bupivacaine hydrochloride as a model drug andmagnesium carbonate powder (fine powder sieved to collect fraction below300 microns in size) as a biocompatible and biodegradable exemplaryfiller and the mixture was vigorously vortexed for 5 minutes. Themagnesium carbonate suspension was infused/tattooed using an oscillatingneedle in 1 cm square area. Excess solution from the tattooed surfacewas wiped off. The light blue tattoo with magnesium particles wasclearly seen the unaided naked eye. In another embodiment, polyglycolicacid (PGA) microparticle is an exemplary biodegradable polymeric filler.In another embodiment, cat gut suture based microparticles (mostlycollagen based) were used as filler. In another embodiment, crosslinkedgelatin or PEG based biodegradable microspheres are used as a fillermaterial. The crosslinking prevents dissolution of the microparticles inthe injectable medium. All the illustrative fillers used were insolublein the organic water soluble solvent used. The insolubility leads tosuspension or emulsion formation. The injected solution precipitates orforms a gel in the aqueous environment present in the tissue. It ishypothesized (invention is not necessarily bound by the hypothesis) thatduring precipitation step, the filler particles provide large surfacearea for polymer precipitation thereby accelerating precipitation andalso provide a larger area for controlled drug release. Filler can alsoalter mechanical properties of the precipitated polymer. As the fillerdissolve or degrade, they can create/alter localized chemicalenvironment such as pH of the surrounding area. The change in localizedpH may also affect the release profile of drug. It is hypothesized(invention is not limited and bound to the hypothesis) that themagnesium carbonate creates a localized mild basic environment which mayconvert drug salts like bupivacaine hydrochloride into bupivacaine basewhich has a much lower water solubility in water than bupivacainehydrochloride salt. This change in drug solubility can potentiallyaffect the sustained release profile of the drug. The basic nature offiller can also neutralize the hydroxyacids created duringbiodegradation of polylactones or polyhydroxy acids such as PLGA. Fillerlike PGA microcylinders or microparticles can produce localized acidicenvironment and may also alter the drug properties during degradationprogress. Irrespective of mechanism of filler action, fillers can beuseful additives for local sustained release of therapeutic drugs whenused with in situ gelation systems like polymer solution in watermiscible organic solvent.

FIG. 21B shows release profile of bupivacaine hydrochloride from the insitu made PLGA array implant with and without magnesium carbonate as anexemplary filler. The filler particles are insoluble organic solvent andhave fine particle size. The data shows that the addition of magnesiumcarbonate has extended the release of bupivacaine hydrochloride from oneto two days to several days. The average size of fillers used may varyfrom 0.1 microns to 500 microns, even more preferably 0.5 microns to 300microns. The preferred inorganic/organic fillers have low watersolubility and produce localized pH around 7.4 (close to physiologicalpH). Salts that can be used as filler include but not limited to:calcium benzoate, calcium citrate tetrahydrate, calcium hydroxide,calcium sulfate, gadolinium(III) sulfate octahydrate, magnesiumcarbonate dihydrate or trihydrate, silver acetate, zinc formatedihydrate, ferrous ammonium sulfate, calcium gluconate, magnesiumtartarate, calcium lactate, calcium tartarate and the like. Thepreferred compounds include inorganic salts and organic salts and saltsof magnesium and calcium metal. Salts of hydroxy acids, mono-acids,di-acids, tri-acids and polyacids are most preferred. Organic salts withC1 to C22 carbons are even more preferred. Salts of organic monoacidssuch as, lactic acid, formic acid, oleic acid, steric acid, acetic acid,propionic acid, butyric acid, valeric acid, caproic acid, benzoic acidand the like may be used. Diacids such as malonic acid, succinic acid,glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid,sebacic acid, maleic acid, fumaric acid, itaconic acids, oxalic acid,aspartic acid, glutamic acid, tartaric acid, terephthalic acid, citricacid, undecanedioic acid, dodecanedioic acid, glutaconic acid, traumaticacid, muconic acid; polyvinyl pyrrolidinone-co-polyacrylic acid,polyacrylic acid copolymers, polyaspartic acid, hyaluronic acid, proteinor peptide sequences comprising two or more acid resides;ethylenediaminetetraacetic acid, methanetetracarboxylic acid,1,2,3,4-butanetetracarboxylic acid, PEG derivatives with acid end groupsand the like. Di or polyacids containing unsaturated groups like fumaricacid, maleic acid, itaconic acid and the like may also be used. Thepreferred salts used must be biodegradable, biocompatible and non-toxicotherwise they cannot be used. One illustrative preferred salt ishydroxy acids such as calcium gluconate, which has a kPa around 6-7.Salts with pKa value around 7 are most preferred. In bone relatedapplications, calcium salt based compositions are preferred. Salts likehydroxy apatite, calcium sulfate, calcium phosphate and the like arepreferred.

Apart from inorganic particulate filler, biodegradable polymer orhydrogel microparticles/microspheres may also be used as filler materialin the in situ gelation systems as described above. Optionally fillermay be stained or encapsulated with visualization agent like coloringagent or fluorescent agent to assist deposition in the tissue. Severalembodiments in this or related application or in cited art providecompositions and methods for preparation of biodegradable polymer orhydrogel microparticles/microspheres. Such methods can preferentially beused to make biodegradable polymer or hydrogelmicroparticles/microspheres for use as filler in this application.Microparticles comprising synthetic biodegradable polymers are mostpreferred. Microparticles comprising PEG based crosslinked hydrogels orPEG-polylactone based polymers are most preferred.

Devices for Making Microarrays Comprising Drug/Cell

In this invention, specialized devices have been disclosed which enablesto implant preformed microimplants or in situ formed microimplants in anarray format. The inventive devices enable to form implanted microarraywithout having sharp cutting edge to the implanted material or withouthaving a backing material. The devices also enable to use materials likesoft hydrogel in hydrated form as array microimplant materials. Theinventive device uses two microneedles arrays (an outer and inner array,also referred as “array in array”) wherein inner array can be insertedin the outer array. The microimplants present in the outer array arepushed out of outer array once the inner array is inserted in outerarray. If the arrays are inserted in the tissues, then the microimplantsin the outer arrays can be inserted in the tissue to form microarray ofimplants.

The inventive devices use arrays made of hollow and/or solidmicroneedles. Materials for hollow microneedle array are made out ofmetal or plastic or ceramic with sharp edges and are capable ofpenetrating the tissue with little pressure or force. The hollow cavityin the needle is used to store/carry the desired drug/cell deliveryimplant. Upon insertion of hollow needle in the skin/tissue at desireddepth, the microimplant in the hollow cavity is pushed out in the tissueand needle is withdrawn from the tissue leaving behind the implant fortherapeutic use. An illustrative “array in array” device is described inFIGS. 6 and 15, more specifically in FIGS. 6A, 6B, 6C, 6D and 15A to15F. FIGS. 6A and 6B show partial schematic representation of “array inarray” apparatus (AIA apparatus) useful in forming microimplant array inthe skin or tissue. The apparatus comprises two parts namely “basearray” or “outer array” with hollow microneedles and “plunger array” or“inner array”, both schematically shown in FIG. 6A and FIG. 6Brespectively. The base array has a base plate with plurality of sharphollow microneedles protruding perpendicular from the surface of thebase array plate. The plunger array (positioned on top of the basearray) also has a plunger plate with a plurality of solid needlesprotruding perpendicular to the plunger plate from its bottom surface.The arrangement, length/size and shape of plunger array and base arrayneedles is identical except the plunger array needle fits smoothlyinside the hollow cavity of base array needle and can move freely insidethe cavity up and down as needed. Panel 6C-1 shows plunger array on topof base array, with center of both corresponding needles are aligned butnot inserted. The spacer lock (6024) prevents the plunger array beinginserted completely. Panel 6C-2 shows plunger array on top of base arrayinserted completely after removal of spacer lock. Plunger array needlesoccupy space in the base array cavity. Panel 6D-1 shows base arraycavities filled with preformed or in situ generated implants (6013) withdrug and/or cells and is ready for implantation. 6013 microimplant couldbe porous and its porosity could be partially or completelyfilled/coated or impregnated with injectable compositions withdrug/cells described in this invention. Pantel 6D-2 shows base array isinserted in the skin tissue and plunger array plunger needles are usedto push the implant in the skin tissue and form an implanted array inthe skin tissue. Both the arrays are removed from the tissue leavingbehind the implanted array with drug/cells in the skin tissue.

“Array in array” (AIA) devices described above have many variations andcan be modified to form different types of arrays. In one illustrativeworking apparatus prototype was prepared according to FIG. 6 and is usedto form microimplant array in the gelatin gel which is used as a modeltissue substrate to conduct laboratory experiments. Use of gelatin helpsto reduce number of animal experiments in designing suitable formulationfor a given drug or cell type. The partial description and images ofworking prototype are given in FIG. 15 for illustration only and doesnot limit in terms of number of needles in the array, size of needles,needle arrangement, volume of cavity, materials used and the like. FIG.15 shows a schematics of a working prototype of illustrative “array inarray” apparatus as described in FIGS. 6A and 6B. FIG. 15A shows basearray (1501) with top view showing 5 by 5 hollow microneedle arraycreated in stainless steel metal plate. Length and breadth of plate is20 mm and thickness is 1 mm. Outside diameter (OD) of the hollowmicroneedles is 0.55 mm while internal diameter (ID) of hollow cavity is0.31 mm. The opening of hollow needles in 5 by 5 format (proximal end ofhollow needle 1502 with cavity ID 0.31 mm) on the plate surface isclearly seen. The base array plate has 4 guiding posts (1503) withdiameter 2.5 mm. Distance between each needle is 2 mm. FIG. 15B showsthe side view of same base array (1501) showing base metal thickness andhollow microneedles (1502) protruding out of the base plate surface. Theouter needle edge (distal end of hollow needle) is cut at 30 degreeangle and cut needle is polished to get sharp edges for ease ofinsertion in the tissue. FIG. 15C shows plunger array (1505) with topview showing 5 by 5 microneedle array with solid plunger needles (1508)created in stainless steel metal. Length and breadth of base plate is 20mm and thickness is 3 mm. Outside diameter (OD) of the solidmicroneedles is 0.3 mm and length is 2 mm. The plunger array plate has 4guiding holes (1507) with diameter 2.6 mm. Both the array needles haveidentical arrangement (5 by 5 array format). Distance between eachneedle is 2 mm. FIG. 15D shows the side view of plunger array 1505showing base metal thickness and solid needles protruding out of thebase plate surface. The solid needles at distal end do not have cuttingedge but a smooth flat surface useful for pushing the implants and theproximal end is attached the plunger array plate.

FIG. 15E shows the plunger array 1505 placed on top of base array 1505(not inserted but aligned and ready for insertion) wherein center ofeach needle of plunger array is aligned with center of base arrayneedle. FIG. 15F is same as FIG. 15E where plunger array needles arecompletely inserted in cavities of base array needles and both arraysbase plates are touching each other. FIG. 15G shows PLGA basedcylindrical microimplant with coumarin as model drug and fluorescentagent is formed in situ inside hollow cavities of base array first andthen pushed inside gelatin gel using plunger array as shown in FIGS. 15Eand 15F. The green fluorescence of implanted array (FIG. 15G, 5 by 5array) under blue light is clearly visible. This shows that the devicecan form biodegradable polymer based microimplants arrays in tissue(gelatin used as model tissue material).

FIG. H shows catgut suture based cylindrical microimplants with coatinginserted in sheep skin tissue in an array format. The preformed coatedcylindrical microimplants (fluorescent coating is on the implant surfacealong the height of the cylinder but not on the base surface) are placedin hollow cavities of base array and then inserted in the sheep skintissue using plunger array as described above. The inserted microimplantshow green fluorescent coating on the outer edge of the implant underblue light. The apparatus used in making arrays (FIGS. 15G and 15F) isone of the several prototypes made and are described in FIGS. 6A, 6B, 6Cand 6D. Base array with metal needles and plastic base plate and plungerarray entirely made of plastic material is preferred.

Example 22A teaches illustrative methods by which the AIA apparatus ismade. The method is given for illustration only and does not limit thisinvention to this example only. Those skilled in the art know that othermethods such as carving the needle and base plate using modern CNCmachines or other tools are possible and may also be used. Lithographicmethods to etch a given pattern in silicon or metal known insemiconductor chip manufacturing process can also be used. Threedimensional manufacturing/printing methods such as methods used insintering of metal/plastic powders or use of melted polymer orphotocrosslinked polymers to form 3 dimensional objects (also known asstereolithography or 3D printing) may also be used. The choice willdepend on cost, ease of manufacturing, acceptable tolerance of each partof the device and other business variables considerations. In oneillustrative embodiment (Example 22, Design 3), a rubber based baseplate is used to provide flexibility to the AIA apparatus. Thisflexibility helps to adjust/conform to skin or tissue surfaces withcurvature. The hollow needles array and plunger needle array may also beinjection molded along with the needles with flexible plastic orthermoplastic elastomer materials as base plate similar to commercialarray shown in FIG. 22A. Flexible metal based band designs such as usedin wrist watch may also be used. The thickness of the base plate (t) maybe 100 microns to 5 mm, preferably 0.5 mm to 3 mm. Size and shape ofbase plate will depend on number of needles, cost and ease of processingand other factors. Base plate shape may be square, circular, triangular,rectangular, square, oval, hexagonal, pentagonal and the like. The areaof the base plate may be 0.1 cm square to 100 square cm, preferably 0.5square cm to 20 square cm. The material of the base plate can beceramic, plastic or metal. Choice will depend on cost, ease ofprocessing, dimensional stability and ease of use and manufacturability.Disposable materials like commonly used thermoplastics likepolyethylene, polypropylene, polyurethanes and the like or thermoplasticelastomers like polyurethanes are preferred. Materials that provide highdimension stability, easy to sterilize are preferred. Commonly usedmedical device materials, particularly materials that are resistant toorganic solvents are preferred. In some cases, the organic solvent maybe used to cast implants in the needles and solvents used may be exposedto the base plate and needle materials. In such case, material shouldwithstand such solvent exposure. The hollow needles may be made out ofceramic, metal or plastic. Plastic or metal are preferred. Chemicallyinert plastics such as polypropylene, polytetrafluoroethylene and thelike are preferred. Metal based needles such as stainless steel needlesare most preferred. If desired, the inside surface of the needle may belubricated with biocompatible lubricants such as vitamin E, Vitamin K,oleic acid, silicone oil, polyethylene glycol, glycerol and the like.The choice will depend on the implant materials being used in the array.The lubrication reduces friction and therefore provides smooth pushingof microimplants, preferably unibody microimplants in the tissue withminimum force. The base array needles design and edge is chosen in suchway that minimum force is needed to push it in the skin or tissue. Theforce required will be different depending on the type of tissue used,number of needles in the array, needle cutting edge design, needlesharpness, angle of force application and the like. Generally base arrayneedles may be able to penetrate the skin tissue with a force per needlein the range of 5 to 50 N/CM², preferably 10 to 20 N/CM². The force maybe further reduced by lubricating the needle external surface withlubricants such as silicone oil or glycerol or other needle lubricantsknown in the art. Preferably the injectable composition ingredientsshould not dissolve or chemically react with the lubricant used. Theinside surface of the needle may be lined or coated with inert ornon-stick material like PTFE for ease of insertion. For hydrophilicmaterials, a hydrogel based surfaces may be more useful. The needlelength for both arrays, base and plunger, may vary from 40 microns to 5mm, preferably 60 microns to 3.5 mm, even more preferably from 100microns to 2 mm. The most preferred length is 100 microns to 1500microns. The thickness of the hollow needle is important. The preferredthickness is the minimum thickness required to penetrate the tissuewithout bending or damaging. The wall thickness of the base hollowneedle can be 10 microns to 1000 microns, preferably 20 microns to 700microns and even more preferably 50 microns to 500 microns. The strongerand harder materials would enable to use needles with minimal wallthickness. Titanium or titanium alloys, Nitinol, aluminium alloys,stainless steel, other cobalt, iron and nickel based alloys and the likemay be used to make thin walled microneedles for the base array. Thebase array hollow microneedle used must have sharp edge at distal endfor smooth tissue penetration. The distal sharp edge which penetratesthe tissue during implantation can have various shapes such as taperpoint, blunt taper point, cutting edge, reverse cutting edge, taper cutand spatula curved and the like. The needle at the distal end (tissuecutting edge) may be cut at 10 to 70 degree angle, preferably 30-45degree angle α. The average internal diameter of base array hollowmicroneedle, d, may vary from 1 micron to 3500 microns, preferably 5microns to 2500 microns, even more preferably 20 microns to 2000microns. The shape of base array microneedle may include but not limitedto straight obelisk, negative-beveled obelisk, cylindrical, pyramidal,conical, trigonal, tetragonal, pentagonal, hexagonal, pyramidal, and thelike or combinations thereof. The pyramid and cylindrical shape ispreferred. The distance between each microneedle in the base array is 1micron to 10000 microns, preferably 3 microns to 3500 microns, even morepreferably 5 microns to 2000 microns. The volume of base array hollowneedle (β) may range 1×10E-12 to 0.05 ml, preferably 1×10E-10 to 0.03ml, even more preferably 1×10E-10 to 0.01 ml. Total number of needlesper array may be greater than 3 or 4 per square centimeters or may rangefrom 3 or 4 to 6000 per square centimeter, even preferably 3 or 4 to1000 and most preferably 3 or 4 to 200 per centimeter square of baseplate area. Total number of needles per array (n) may range from 3 to10000, preferably 3 to 1000 even more preferably 4 to 300. Thearrangement of microneedles in the array is preferred like a matrixwhich has m number of rows and n number of columns. Each needle in thearray can be identified as respective number of rows and column. Theplunger array also has a base plate and solid needles designed to holdor push the microimplants from the base array needle. The materials usedin plunger array base plate and its size are similar to base array plateas described above. The number of needles in plunger array (n′) may varybut must be equal to or less than base array needles (n). If n equals oris less than n′, then the position of plunger array and base plateneedles on respective arrays must match with each other so that plungerarray needles can be inserted inside the cavities of base array needlesat the same time. Number of needles (n′) can be same or equal to numberof needles in one column or row. In illustrative array such as shown inFIG. 6 or 15, the number of needles in one row or column is five (C1 toC5 or R1 to R5) When such arrangement exists, then plunger array needlescan be inserted in each column or each row to push the implants out perrow or column at a time in the tissue. It is preferred that number ofneedles of base array, n, is equal to number of plunger array needles.The purpose of plunger array microneedles is to push the implant out ofbase array cavity. It generally has smooth surface without sharp edge/sat distal end and should not be able to pierce/tear the in situ formedimplant or preformed implant present in the base array needle cavity.The materials used could be metal, plastic, glass, rubber/elastomeric orceramic, but soft plastic material with flat surface is preferred. Theaverage diameter/size of plunger array needle is less than thediameter/size of base array hollow cavity diameter (e′ is less than d).This enables insertion of plunger array needles in the cavity of basearray needles. It is preferred that it is a tight fit with smooth up anddown movement of plunger array needle in the cavity of base arrayneedle. The value of e′ could be 1 to 80 percent less than the value ofd, preferably 5 to 60 percent less. The length of the plunger arrayneedle (b′) is generally same as length of base array needle (b).

The length b′ could be smaller or larger than the base array needle. Theneedle length b′ could be larger and in some cases, it can be 1.1 to 6times longer, preferably 2 to 3 times longer than base needle. In oneembodiment, 3 times longer plunger needle array is used to remove theimplant from the AIA apparatus in the skin tissue. The base array of AIAapparatus with preformed implant in the hollow cavity is first insertedin the skin tissue (Panel 6D-1). The plunger array with 3 times thelength of base array is then inserted in the hollow array cavities untilthe plunger array needles touch the microimplants top surface in thebase array cavity. The plunger array is held in that position and basearray is lifted up along the length of the plunger array needle and outof the tissue. The extra length of plunger array needle enables thisupward movement. The plunger array is also removed from the tissueleaving behind the implant in the skin tissue. In this method, theimplant is not pushed but is held in place by the plunger array in thetissue while the base array is being removed from the skin tissue. Theplunger array holds the implants in place in the tissue while base arrayis completely removed from the tissue. About three times the length ofplunger provides sufficient space along the axis of the array needle tobe removed from the tissue and thus it enables to move the base arrayneedles out of skin surface. A combination of pushing or holding asdescribed in any proportion may be used to insert the microimplant intissue.

Array needles may have caps or packaging features that helps them toprotect the needles during normal handling and storage of the AIAdevice. Since the device features are small, the package must protect itfrom dust and other particulate matter which can get inside the needlecavities and can potentially block the passages. The needles on botharrays can be bent or damaged during manufacturing and transportationoperation. Specialized protective coverings or caps may be designed toprotect the needles from such damages. The protection may especially beneeded for sharp edged needle array to maintain sharpness for tissueinsertion. The protective covering may be removed easily at the time ofuse.

In some embodiments, the plunger or inner array needle has a passage ortube (6022) along the axis of the needle length and injection port(6023). The purpose of passage and port is to use the plunger array todeliver injectable compositions (polymer solutions, crosslinkableprecursors compositions, thermosensitive gel based compositions and thelike) comprising drugs/cells in the tissue or in the cavity of basearray needle. The average diameter of passage tube/opening is 10 to 70percent of diameter of the plunger needle, preferably 15 to 60 percentand even more preferably 20 to 50 percent. The passage and port may alsobe used to apply air or other gas pressure to push the implant in basearray cavity. Any biocompatible gas can be used but biocompatible gasessuch as carbon dioxide or oxygen are preferred. It can also be used toinject biocompatible liquids (which may be pressurized) such as PBSsolution, saline solution and the like to push the implant in thetissue. The injectable port may be like a Luer lock type port wheresyringe or catheter with injectable compositions can be connected andinjected in the tissue via passage 6022 into in the cavity space βInjectable port may also house microelectronic accessary and sensorswhich enable movement of plunger array needle up and down so that eachneedle array movement can be controlled by a machine or computerprogram. If movement of individual needle is controlled by a machine,then it is possible to choose a desired pattern of implanted array inthe tissue. In one illustrative embodiment, a syringe needle connectedwith pressurized nitrogen gas is inserted manually in each cavity of thearray (base array with prefabricated implant, FIG. 15B). The pressuredgas pushes the implant from the base array needle cavity in to thetissue. The manual pushing of microimplants, one at a time, canpotentially give more control over drug dose to be given perhuman/animal subject but may be time consuming and susceptible to humanerrors.

The AIA apparatus has features that ensure alignment of array needles.This alignment ensures entrance of all the plunger array needles in thebase array cavities at the same time. In one illustrative embodiment,guideposts and guide holes are used as an alignment feature. The baseplate of the base array has four guide posts (FIG. 6) and plunger arraybase plate have 4 holes (FIG. 6) at corresponding location and the sizeof holes is greater than size of guiding posts. The guide posts alsohelp to hold/handle the array by hand during insertion in the skintissue. It is necessary that all the centers of needles of the plungerarray must be aligned with centers of base array needle cavity/openings(opening on the base plate surface). If plunger array needles are notproperly aligned, they may potentially get damaged or bent duringimproper insertion and therefore cannot function to push microimplantsin the tissue. The guide posts help to achieve this alignment. The basearray and plunger array may also have additional markings on theirsurfaces such as arrow/s or line/s or number/s that assists in properorientation while inserting plunger array. Alternatively, guide postsmay be on added on plunger array and holes may be added on base array,however, guideposts on the base array are preferred. The use of guidepost is optional but alignment feature on AIA device is believed to behelpful to the user. Other methods of alignments or other alignmentfeatures such as laser based instrumentation known in themedical/automobile instrumentation art, electronic or mechanical methodsknown in the engineering art may also be used. The alignment of botharray needles is more important than how it is achieved. In oneillustrative embodiment 4 guide posts and holes have been used. Numberof guide posts and corresponding holes could be one, two, three, four ormore depending on the space available, cost and other variables.

FIG. 25 shows partial schematic representation method to make base orplunger array as described in FIG. 6. This is one of the several methodsthat can be used to make the AIA device. Metal hollow tubes with adesired cavity diameter (ID) and wall thickness and length are provided(2501). Hypodermic needles are normally made from a stainless-steeltubes, which pass through a process known as tube drawing where the tubeis drawn through progressively smaller dies to make a tube of desiredsize. The tubes are encased in a plastic or metal plate (2502) via insitu casting of plastic resin or injection molding or wielding/adhesivebonding or other methods. The encased tubes are cut on the base platesurface (proximal end, straight cut to create a tube cavity opening onthe surface) and also cut at distal end and beveled to create a sharppointed tip letting the needle easily penetrate the skin. Standardbevel, medium short bevel or short bevel may be used to form a cuttingedge. The angular cut at desired angle at distal end produces sharpedged (2503) hollow microneedles at distal end. The cut needle edges maybe electropolished to make the cutting surface smooth. The sharp-edgedmicroneedles protrude from the base plate (2501). The cut edges may bepolished to produce a sharp edge. The opening on base plate surface(proximal end, not shown) is used for insertion of microimplant. Thehollow tubes may be substituted with solid rods to produce plungerarray. A laser beam may also be used to cut the tubes and produce sharpedged needle in combination with polishing or electro polishing.Alternatively, base and plunger array, preferably plunger array can beentirely made by injection molding of commonly used medicalthermoplastics. Alternatively, the needles may be first cut to desiredlength with sharp end and then encased in a plastic/metal/ceramic baseplate (2502) to produce needles with sharp edges at distal end andopening in proximal end.

Base array alone or in combination with plunger array is used to formmicroimplant array in the skin or tissue. The apparatus as describedabove may be used to form implant in situ inside the tissue or may beable to inject a preformed implant with well-defined shape and size anddrug release profile may be used. The preferred microimplant (eitherformed in situ or preformed outside in a factory setting) has avisualization agent. The preferred visualization agent is abiocompatible and biodegradable colored or fluorescent compound. Thecolor or fluorescence of the injectable composition or implant helps tosee the array during and after implantation procedure. In one exemplaryembodiment (Example 22B), the AIA array is used to form in situ implantin a gelatin gel which can be used as a model tissue substrate becauseit is transparent in nature and helps to optimize implantationconditions and compositions. 5-15 percent gelatin solution is cast into4 mm thick 1 inch diameter gel. The array in “array in array” devicesimilar to device described in FIG. 6 or 15 is used. The base arraydevice such as shown in FIG. 15B and plunger array plunger device suchas shown in FIG. 15D are used. The plunger array (PA array) with 25needles is inserted in the base array corresponding base array cavities(such as shown in FIG. 15F). In this arrangement, most of the hollowcavity space in the base array needles is occupied by the plunger arrayneedles. The device (similar to shown in FIG. 15F) is inserted ingelatin gel or skin to create 5 by 5 array holes. The tissue/gelatin gelcannot enter in the cavity space of base array needles because the spaceis preoccupied by the plunger array needles. Upon complete insertion,the plunger array is removed from the base array and the space/volumecreated by the removal of plunger array microneedles is then filled byan injectable composition. Briefly, 1 g of PLGA (50:50lactide:glycolide, PDLG 5002) polymer and 10 mg of coumarin and 9 mln-methyl pyrrolidone are mixed until complete solution. The greensolution is injected using a syringe in each base array needle cavity(B, 25 total cavities). After filling cavities, excess solution is wipedoff. The cavity is exposed to 2 ml PBS solution to accelerateprecipitation of the polymer in the cavities. Using a plastic rod withsize less than size of the hollow needle cavity, each precipitatedmicroimplant is individually pushed into gelatin gel. Alternatively,plunger array is inserted to push all implants at once. The advantage ofindividual insertion of implant is that the number of implants and thearray design/arrangement can be controlled.

Generally, the drug amount per microimplant per array needle is verysmall and each implant can be individually inserted, enabling to deliversmall dose per microimplant. If needed, the total dose can be furtherincreased by inserting additional microimplants from the array. Also, itmay be possible to leave certain position in the matrix unimplanted andsuch positions can be used to encode certain information. The locationthe implant where it is present is encoded as one and the locationwithout the implant is encoded as zero. The combination of zero and onecan be used in encoding information similar to the used in moderncomputers. Another advantage of individual insertion of implant in anarray is that the pattern implanted array can be chosen for a givenmedical need. For example, if needed, the implants in the first row(R1C1, R1C2, R1C3, R1C4 and R1C5 per FIG. 6A) can be inserted in thetissue out of 25 implants in the 5 by 5 format (FIG. 6A). If amicroimplant in R1C1 can deliver a drug for one day, then each implantin the array (25 total implants in 5 by 5 array) can be programmed to beinserted per day. This way 25 day sustained drug delivery can bemanaged. The programming can be machine controlled or can be donemanually. In another embodiment, if each microimplant in the array candeliver a drug for one week, then the entire array can be used todeliver 25 weeks sustained drug delivery. It is preferred that theneedles are in the tissue for a short period of time during implantationtime only, otherwise they can be on the skin/tissue surface but notinserted. Thus, by controlling variable like number of microimplants inthe array, amount of drug per implant, duration of delivery per implant,insertion of number of implants per day and the like a sustained drugdelivery composition, its dose and its duration can be controlled usingthis device.

The gelatin gel with PLGA microimplants is cut into rectangular shapeand is photographed under blue light (FIG. 15G). The 5 by 5 array ofPLGA based microimplants is clearly visible in gelatin gel and isfluorescent in nature (FIG. 15G). The use of polymer solution in watermiscible solvent is used as illustrative injectable composition. Otherinjectable compositions described in this invention may also be used.The list of biodegradable polymers, biostable polymers, solvents forpolymers, list of drugs that can be used is described elsewhere. Inanother exemplary embodiment (Example 22B), the AIA device is such asshown in Panel 6C-2 or FIG. 15F (plunger array is completely inserted inbase array cavities) is inserted in the porcine skin tissue to create 5by 5 array holes. The presence of plunger array needles in the hollowneedles prevents the tissue coring or insertion in the hollow tissuecavity of base array. The plunger array is removed from the base arrayand the created space in the base array needle cavity is filled withinjectable composition comprising live cell suspension and crosslinkableprecursors solution such as fibrin glue or macromonomer solution(Example 15 and Example 10D). The precursor solution is crosslinked insitu encapsulating live cells in the crosslinked gels. The formedmicroimplants take the shape of base array cavity. The formed implantsare then pushed inside the skin tissue with the plunger array thusforming microimplant arrays with live cells in the skin tissue.

In another embodiment and modification of AIA device, one microimplantat a time is pushed or injected in the tissue from base array cavityusing a mechanism similar to used in firing bullet from a pistol orrevolver. Generally, revolver/pistol is a repeating handgun that has arevolving cylinder containing multiple chambers and at least one barrelfor firing. The revolver enables the user to fire multiple roundswithout reloading. Each time the user cocks the hammer, the cylinderrevolves to align the next chamber and round with the hammer and barrel.A similar mechanism can be used to fire/push implant wherein thechambers of barrel (cavities in base array) are used to storemicroimplant and the firing/pushing is done by plunger of the arrayneedle or similar mechanism. In some cases, the tissue used for cavitymaking has a tendency to recoil due to elastic nature of the tissue. Therecoiling may change or reduce the size/volume of the cavity or closethe entrance of the cavity. In such circumstances, the cavity createdinside the device (AIA device described in this invention) may be usedor the composition may be injected prior to elastic response. Forexample, the hollow microneedle hollow needle array used in someembodiments (33 MP array) can be inserted in the tissue and thenwithdrawn from the disuse. During the partial needle withdrawal, theinjectable composition can be injected before the tissue recoils in thecavity created by needle.

Alternatively, tissue destruction can be used in place of tissuedisplacement. Those skilled in the art will realize that the choice ofmethod will depend on the medical need, cost, ease of use and othervariables.

In another embodiment of the AIA device, use of expandable array needlein forming drug delivery microimplant array has been shown. The needleof the AIA device (base and/or plunger, preferably plunger array) can beexpandable. FIG. 23 shows schematic representation of use of expandablearray needle in forming drug delivery microimplant array. 2601 is anexpandable needle/stent with hollow cavity for storage of drug/cellcomprising microimplant. The needle is present in the compact form inthe base array cavity needle in AIA device. The base array cavity spaceprevents the expandable plunger array needle from expansion. FIG. 23Ashows an expandable needle in compact form with microimplant (6013) inits cavity and is pushed out from the base array cavity into the skintissue in unexpanded form but with implant in the cavity. FIG. 23B showsthe expansion of plunger array needle/stent into an expanded shape or toits memorized shape (2302). The expanded shape has been pre-memorizedinto needle/stent using a heat treatment of the Nitinol alloy. Theexpanded shape releases the implant in the skin tissue and is thenwithdrawn in the base cavity array in compact form and then out of theskin tissue (FIG. 23C). The microimplant (6013) is left in the tissue inan array format for therapeutic action. The use of Nitinol based shapedmemory alloys for making stent like devices is well known in stent basedmedical device art. The Plunger array needle device can be made usingNitinol alloy. The needle of the plunger array has two shapes. A compactshape and an expanded shape. Compact shape (2601) has cavity for storageof drug delivery microimplant. The Expanded shape such as conical shape(2602) is memorized into Nitinol alloy by heat treatment. The Plungerarray with microimplants in its cavity is inserted in the base arraycavity in compact form. The base array cavity's limited space preventsthe expansion of compact form to expanded form at room temperature orbody temperature. When base array and plunger array is inserted in thetissue, the plunger array needle is pushed out of base array needlecavity and body temperature causes the plunger array needle to expandand release the drug delivery microimplant in the tissue (FIG. 23B). Theplunger array is pulled back in to base array cavity and then out ofskin tissue leaving behind the drug delivery implant for therapeuticaction. The use of Nitinol materials for expandable needle is used forillustration only and is not a limitation to this invention. Otherexpandable designs such as known in the stent medical device art likeballoon expandable stent may also be used. The micro-balloon suited forthe expandable needle may need to be specifically designed for theneedle expansion and then used.

The AIA apparatus also can be used to implant preformed microimplantsmade in a factory setting. The microimplants can also be made outsideusing well established polymer processing techniques like extrusion,injection molding, solution casting, sintering and the like. In oneembodiment, microcylindrical implants coated with drug deliverycompositions are formed. The size and shape of the implant is chosen insuch way that they can fit inside the cavity of AIA base array needlesor inside cavity of plunger array expandable needle. In one embodiment,cat gut based fibers/threads are first coated with PLGA and coumarinbased compositions as described before to obtain a coated fiber. Thecoated fiber is then cut into several microcylindrical rods ormicroimplants. Average length of cut microcylinder is 496 microns andPLGA coating has a thickness of approximately 40 microns (FIG. 15H). Thecut microcylindrical rods are placed inside the cavity of base array.The plunger array is placed on top of base array with 2 mm polyethylenesheet as a spacer lock (6024). The device is then transported on top ofporcine/sheep skin and bottom array needles are inserted completely inthe skin. The spacer sheet is removed and the plunger array is pushed inthe base array cavities. The plunger array pushes the implant in theskin. Both base array and plunger array are removed from the skin tissueleaving 25 implanted rods in array format. The implanted array canprovide sustained drug delivery and fluorescent coating helps tovisualize the implants in the skin. FIG. 15H shows coatedmicrocylindrical rods in the skin tissue imaged under blue light. Theimplants did not have sharp cutting edge but can be implanted in thetissue using AIA apparatus. The microneedles of base array can do thecutting and insertion in the function. The illustrative FIG. 15H imageshows 5 by 5 matrix type implantation arrangement in the skin tissuewith fluorescent green coating. In some cases, a pressurized saline isused to push the implant from the base array cavities in the tissue. Theillustrative array shows only cylindrical implant. The shape of theimplant can vary and could be pyramidal, hexagonal and the like, as longas the size of the implant can fit inside cavity of base array. Thesterile saline solution is attached to 22 gauge needle and 0.5-10 psipressurized saline is discharged from the 22 gauge needle in the basearray cavity to dislodge the implant from the array into tissue. In somecases, pressurized gas such as air, nitrogen, oxygen or carbon dioxideis used in place of saline to push the implant. In one exemplaryembodiment, a 22 gauge needle is connected to carbon dioxide cylinderand preformed cylindrical implants is pushed using the pressurized fluidcoming out of 22-gauge needle. Conical shape implants with sharp needleedge (Example 8, 16, 20 FIG. 9B2) may also be used as prefabricatedimplants (Panel 6A-3-2, 6013). Such implants are useful to push insidethe tissue when pushed by air or gas pressure or plunger. Conical shapeimplants with sharp needle edge made from biodegradable polyester likePLGA can be useful in some applications.

The AIA device and its methods of use enables to deliver the drug in asolid form without forming drug solution or suspension. This can haveseveral advantages over injectable drugs that are made in to solutionprior to use. Table 3 compares the delivery of drug in a solid state asdescribed in this invention and drug injection such as Botox injection.

TABLE 3 Comparison of conventional injectable drug solutionadministrated as a solution and delivering the same using microimplantarray as described in this invention. Conventional liquid/ Drugsadministrated as solutions injectable solid microimplant array asdescribed in this invention. Generally, drug is provided No need toprovide liquid as a sterile solid along diluent. Drug is provided withits specially designed as a solid in the form of liquid diluent (waterfor microimplant array. injection as an example). The diluent must bemeasured, Not applicable. Reduced mixed in a sterile manner. chance oferrors due to Chance of human error in elimination of human steps.measurement or accidental needle pricks or loss of sterility duringpreparation or measurement. Requires a sterile syringe Requires AIAdevice for and needle for administration. administration. Requiresproper storage and Requires proper storage disposal of used syringe andand disposal of AIA device. needles. Distribution of drug in tissuePotential for visualization generally cannot be visually of administeredmicroimplant seen. array. Can be painful. Potential for pain freeapplication. Generally, treatment cannot In some cases, potential bereversed once injected. to remove/denature/destroy the microimplantsafter administration if administered under the skin. Injected liquid canhave Tissue contact area of the different tissue contact area implantedarray is generally of tissue contact. well defined. Injection area isgenerally Injection area can be large limited. depending on the arraydesign used.

In one embodiment, collagen foam based fluorescent microimplants(fluorscein covalently linked collagen) containing 0.1 unit of Botox permicroimplant are made first and then filled in the base array cavities(25 microimplants in 5 by 5 array format) and then pushed in the skintissue as above using a plunger array and both arrays are removedleaving behind collagen based implant with Botox. The array provides 2.5units of Botox per array in the skin tissue. The blue light exposure ofskin tissue shows green fluorescence of collagen indicating successfulimplantation. If the length of base array needle is less than 600microns, it could be a relatively pain free procedure for Botoxdelivery. This method also delivers Botox drug in the solid state(lyophilized state) without dilution with saline. Collagen is used as anillustrative biocompatible biodegradable bulking material in a solidstate. The bulking agent provides bulk or volume to the Botox drug (anillustrative solid state drug) to form a microimplant with unibodyproperties or unibody microimplant with desirable mechanical integritywhich can tolerate routine processing and device based implantationprocess. Other biocompatible and biodegradable natural and syntheticmaterials such as albumin (human or bovine), sugar of various types usedin making dissolvable microarray implants, hyaluronic acid, polyvinylpyrrolidinone, polyethylene glycol, polyvinyl alcohol, hydroxymethylcellulose, hydroxypropyl cellulose, biodegradable polymers likepolylactones or combination thereof and the like may be used as acarrier for Botox drug. Botox drug is used for illustration only, thesame technique can be applied to deliver other drugs or vaccines whichcan be delivered in the solid state form without making a liquidsolution. If the drug has high enough molecular weight, and itsamount/mass used is sufficient to form a unibody microimplant, then itmay not need bulking agent as described before. Botox has high molecularweight but is used in extremely small amount per vial, which may not besufficient to form a unibody microimplant of desired size, thereforehuman serum albumin or hyaluronic acid or collagen is used as a bulkingagent to provide unibody implant properties. For many small molecularweight compounds like rifampin or bupivacaine, or for drug encapsulatedmicrospheres, a bulking agent or binding agent may be needed to form aunibody microimplant structure which then can be implanted in artificialtissue cavities or implanted using AIA device. In some cases, thebulking agent is first made into appropriate size microimplant and theimplant is impregnated with drug solution prior to implantation or itmay also be impregnated in factory setting and solvent is removed. Ifusing just prior to implantation, the care is taken that the drugsolution will preserve unibody properties of the implant. In oneillustrative example, glutaraldehyde or EDC crosslinked collagen orgelatin foam is cut or fabricated into microcylindrical implants, loadedin AIA device cavities. The foam is then impregnated with 1-5 microliterof Botox solution having 0.1 unit of Botox drug is added on the implant.The implant does not lose its unibody structure with the addition ofsmall drug solution. Crosslinked structure helps in maintaining unibodyproperties and prevent dissolution. The Botox impregnated implant isthen implanted in the tissue as described before. In many cases, themicroimplant is produced with the protein drug in factory setting andthen implanted in an array format using AIA device as described before.This is done by mixing the drug and bulking agent in aqueous buffer orsolvent to form a solution or suspension, filling the solution in a moldof desired size and lyophilizing/evaporating to remove water/solvent andto form unibody microimplant with drug. The unibody implant is then usedto form microarray implant in the skin/tissue. In many cases, thesolution is added in the base array cavities, centrifuged for uniformfilling and lyophilized/evaporated the solvent in a factory setting.Alternatively, bulking agent and drug are formed into a solid sheet likematerial of desired size (10 cm length by 10 cm width and 500 micronsthick as an example). The microimplant is then cut/stamped out (300microns size diameter and 500 microns height cylindrical implant as anexample) and then deployed in artificial tissue cavities or deployedusing AIA device to form a microimplant array in the skin/tissue asdescribed before.

In another illustrative embodiment, according to the “array in array”arrangement where the device is inserted in the skin/tissue in closedposition (such as illustrated in FIG. 15F) wherein the plunger array orinner needle substantially or completely occupy the space inside outerneedle or base array needle. Tissue/skin insertion in a closed positionprevents tissue from going inside (tissue coring) the base array needlecavity during insertion step because most of the cavity volume ispreoccupied by the plunger needle. The inner needle has a relativelysmall opening to inject polymer solutions or other injectablecomposition and it does not affect the ability of tissue to go insidethe needle. The sharp edge of the outer/base array needle enables easypenetration inside the tissue. Once inside, the inner/plungertube/needle is pulled up like a piston moving inside a tube, whichcreates a cavity in the base array needle. The cavity is then filledusing the passage or tube present inside the inner needle. The innertube (6022) which pushes injectable fluid, may be multilumen tubing sothat two different liquids can be injected in the cavity via each lumen.This could be useful to add compositions like fibrin glue whoseprecursors may be added inside the cavity and each precursor may beadded via each lumen present in the tube. The injectable compositionoccupies the cavity, partially (10 to 90 percent) or completely. Theinjectable composition undergoes chemical or physical transitions and“sets” inside the cavity. The outer device can be withdrawn from thetissue leaving behind the set polymer. For example, a low meltingpolymer can be melted and injected in the cavity and cooled to formsolid implant. Crosslinkable precursors like fibrin glue precursors orprecursors present in DuraSeal surgical sealant may be added and allowedto crosslink and form a biodegradable synthetic crosslinked gel.Injectable composition could be polymer dissolved in water miscibleorganic solvent and solvent is allowed to dissipate inside the tissue,which leads to polymer precipitation and forming a solid implant.Prefabricated microimplants with drug/cells may also be loaded in devicecavities and then pushed into skin tissue. The FIGS. 6A-6D and 15A-Hshow a cylindrical cavity being formed inside the device, but it couldbe of any shape, preferably symmetrical shape, as long as the inner andouter tube can be removed without affecting the set implant. The shapecould be cubical, cuboid, prism and the like.

The hollow needle used for deployment of implants should not have narrowopening at the distal end (d1 is less than d) because upon casting, thecast implant cannot be pushed out from the narrow opening. This is alsotrue for deployment of solid prefabricated implant through the device.The average diameter of cast implant or prefabricated implant must besmaller than the smallest of the needle diameter (d or d1). If d1 issmaller than d, the cast or prefabricated will require excessive forceto squeeze out through the narrow opening and such situations should beavoided. It will also impede the removal of needle from the tissue andcan pull back the cast implant with it. The average internal diameter ofhollow needle (d) is preferably less than or equal to d1, except in caseof expandable needle shape described elsewhere. Those skilled in the artcan understand the choice of shape and size of the prefabricatedmicroimplant or in situ case implant must be chosen such that it can beeasily pushed out from the distal end of the needle. Those skilled inthe art understand that needle shape and size must be chosen in a waythat it should not hinder the removal of device needles from theskin/tissue. Those skilled in the art recognize that many variations arepossible with the illustrative embodiments presented herein. Variablelike needle shape; needle internal diameter (d and d1); needle externaldiameter; spacing between each needle in the array; needle materialtype;

number of needles per array; number of array insertion points and thelike may be used to make variety of microimplant arrays in the tissue.The embodiments illustrated and described herein may be shown to have anarrowed opening relative to the lumen of the hollow microneedle,however, any of these embodiments should be understood to also allow forthe entirety of the lumen of the hollow microneedle and outlet at thetip to have the same cross-sectional profile and cross-sectionaldimensions in order for a solid microimplant within the lumen to becapable of being pushed out of the lumen by the plunger shaft (e.g.,solid microneedle that is blunt tipped.). Accordingly, during removal ofthe microneedle, the microimplent will be retained in the medium (e.g.,live tissue or bioprosthesis tissue). However, if the distal opening atthe tip of the microneedle is narrowed relative to the shaft lumen, thecomposition may be malleable or compressible so that it may be pushedout the narrowed opening during or after withdrawal of the microneedlefrom the medium so that the formed cavity receives the microimplanttherein while allowing withdrawal of the microneedle from the medium.

In another modification of AIA device, a separate cartilage containingunibody microimplants is made and used. The cartridge is either shippedwith an AIA device as a kit or it may be used as a single packagecomprising base array, plunger array and microimplant cartridge. Thecartridge is specifically designed to work for a specific AIA apparatusdesign. If AIA design changes, then cartilage must be redesigned to workwith modified redesigned AIA device. FIG. 24 shows a partial schematicrepresentation of illustrative “array in array” device wherein aseparate cartridge for holding microimplants is used instead of space inhollow cavity of the base array. The cartridge can be aligned and placedbetween base outer array and plunger or inner array. The microimplantsin the cartridge are then inserted into skin/tissue via base arraycavities. FIG. 24, item A1 shows an illustrative circular shapedexemplary cartridge. The cartridge has a base plate (2401) and has oneor more holes/cavities (2402). The holes have openings on both sides ofthe cartridge plate surface 2401 (proximal and distal end). The holes2402 can be filled with preformed or in situ formed microimplants (6013)with drug/cells (FIG. 24 item A2). Optionally bottom and/or top surfaceof 2401 may be covered with protective cover (2407) which may be removedat the time of use or it may implantable and/or biodegradable. Theprotective cover prevents unwanted movements of implant from the holes.FIG. 24 item B1 and FIG. 24 item B2 represent base array and plungerarray respectively similar to described in FIGS. 6A-6D wherein 2405 is abase plate to which hollow sharp needles are attached and proximal endof needles has opening on the base plate to load microimplants. FIG. 24item B2 is similar to the plunger array described in FIGS. 6A-6D wherein2405 is a plunger plate to which solid non-cutting needles (2406) areattached. The internal diameter of hollow needles (2404) is same as holediameter in cartridge FIG. 24 item A1 (2402). The external diameter ofplunger needles (2406) is less than the diameter of holes in FIG. 24item A1 and hollow needles in FIG. 24 item B1 and it can freely move upand down in the holes/cavities of FIG. 24 item A1 and FIG. 24 item B2.The number of needles and holes and their size, shape and arrangement inthe array is identical in FIG. 24 items A1, B1 and B2. FIG. 24 item Cshows array B inserted in the skin tissue and cartridge A2 withmicroimplant is placed on top of array B1 with protective cover 2407removed. The center of all the holes in FIG. 24 item A1 is aligned thewith the center of hollow needle opening on base array plate FIG. 24item B1. The center of plunger array needles in FIG. 24 item C is alsoaligned with center of holes in FIG. 24 item A2 and FIG. 24 item B2 butis not inserted in cartridge A2. FIG. 24 item D shows the insertion ofplunger needles in the holes of cartridge A (2402) and cavities of B1(2404) and pushing the implants from them in the skin tissue. Both thearrays and cartridge are pulled from skin tissue leaving behind themicroimplant array with drug/cells for local or systemic therapeuticeffect. The cartridge may be packaged and stored separately and used asdescribed above or it may be packaged in the pre-aligned form in the AIAdevice and used for implantation. The cartridge, plunger array may haveadditional holes (not shown) and base array may have guiding posts (notshown) to help in alignment similar to described in FIGS. 6A-6D. Adrug/cell loaded unibody microimplant may be manufactured, sterilizedand packaged separately than the AIA device or it may be pre-inserted,pre-aligned and deployed along with AIA device. In one illustrativeembodiment (Example 22, Design 4) a cartridge that has a base plate withholes to store drug/cell implants and optionally a protective coveringon top and bottom plate to prevent movement of implant during storageand handling is made. A stainless steel rectangular plate 500 micronsthick and 20 mm length and 20 mm width is used. At the center of theplate 25 holes with 0.29 mm diameter are drilled in the 5 by 5 matrixformat. The matrix position and format is same as base array needleformat as shown in Example 22, design 2 or array used in FIGS. 15A-15H.The plate is kept on Teflon sheet and the holes of the plate are filledwith PEG based macromonomer solution with photoinitiator (Example 10)and bupivacaine loaded PLGA microspheres (20 percent loading). Foraqueous solutions, hydrophobic surface like Teflon surface preventsdroplet spreading which helps to stay the solution inside the cavity.The monomer solution/suspension is exposed to long UV light (360 nm).The polymerization forms biodegradable hydrogels inside the holes withdrug loaded microspheres. The water in the hydrogel may be removed bylyophilization. A protective polyester sheet is placed at the bottom ofcartridge plate. The plate with solid lyophilized microimplants withdiameter 0.29 diameter is placed on top of base array plate with centerof holes matching the center of base array cavity holes and theprotective cover is removed. The base array needles are then inserted inthe sheep skin tissue and the plunger array is kept on top of cartridgearray where center of plunger needles aligns the center of cartridgeimplant. The plunger array is pushed downward which pushes the hydrogelimplant via base cavity array into tissue. The base array along withcartridge and plunger array are removed from the tissue leaving thehydrogel implant for local or systemic drug therapy. The stainless-steelplate of cartridge can have holes for alignment which can be used withguiding posts on the base array surface. In another variation,prefabricated cylindrical implants such as shown in FIG. 15H are used.To hold the implant in place, the base plate bottom surface has beenspray/dip coated with collagen or gelatin film foam or other filmforming biodegradable biocompatible polymer. The part of the protectivecovering may be made from water soluble and/or biodegradablebiocompatible polymer. The implants are pushed and the coated film/foamis removed just prior to use. In another embodiment same as above, theprotective foam cover is not removed but is cut/stamped out by theplunger array and is transported inside the tissue and implanted (inthis case plunger array needles have a cutting edge to stamp out thefoam). The gelatin/collagen is safely removed by the body bybiodegradation process. In another embodiment, biodegradable polymerimplant (PLGA implant) is cast from a solution in situ, solvent isremoved and the microimplant is inserted in the tissue as discussedbefore. In another variation, stainless steel base plate in thecartridge is substituted with silicone rubber sheet with holes for guideposts for alignment. In another variation, a biodegradable foam such ascollagen foam with same size as base plate of cartridge base plate (500micron thick) that is infused with drug/cells is used as a cartridge.The foam is placed on top of base array plate and a plunger array isinserted in base array cavities via foam. The plunger array needlescut/stamp the foam and the cut foam portion of the foam is pushed viabase array needle cavity into skin/tissue as described before. In thisembodiment, plunger array needles distal end is designed such that itcan easily stamp out the foam material with little pressure or force.

Oscillating plunger needle that implants/pushes one microimplant perinsertion at 30-120-degree angle, preferably 90-degree angle fromcartridge is also envisioned. The implants are inserted at frequency of1-200 implant per minute from the cartridge via base array needle intoskin/tissue. Each implant is inserted in predetermined new location atpredetermined depth on the skin to form a microarray of desired shapeand size. The micro implants will be loaded in a cassette likearrangement wherein one implant will be loaded from the cassette intooscillating needle prior to skin insertion. The cartridge or base cavityholes may be filled with biodegradable polymer solution with drug,degassed and optionally centrifuged to fill uniformly. The solvent isremoved by evaporation or by lyophilization or other means leavingbehind the biodegradable polymer and drug in the cartridge or base arraycavity. The molecular weight of the polymer is and other properties arechosen such that the implant formed has unibody structure. The unibodyimplant is then inserted in the skin/tissue as discussed before forlocal or systemic drug therapy.

Number of microimplants that can be formed with AIA device include butare not limited to 1, 2, 3, 4, 5, 6 and up to 100 per square centimeteror higher. The implant formed is biodegradable and preferably containsdrug/cells and/or visualization agent. The device materials could bemetal, polymers or plastics or ceramics or glass. The most preferredmaterials are stainless steel, titanium, gold, silver, polyethylene,polypropylene, nylon, polytetrafluoroethylene or their alloys andcombinations thereof. Among these stainless steel is the most preferred.The device materials should be compatible with solvents and othercomponents in the injectable composition; for example, DMSO used ininjectable compositions can dissolve common plastics like polymethylmethacrylate and thus such materials are not preferred as devicecomponents. Materials described above generally work for aqueous basednon-corrosive compositions.

Another advantage of this inventive method is that there is no need forsharp edges for microimplant materials to be implanted. The base arraymaterial needle can serve the function of tissue insertion. Inconventional biodegradable or dissolvable microneedle for drug delivery,the needle must have sharp edges for easy insertion in the tissue alongwith its backing material and the same material is used as a carrier ofdrugs. The conventional array needle serves as a tissue penetrationdevice as well as it provides sustained drug release properties for thedrug. This requirement of needle sharpness and mechanical strength fortissue penetration with small force limits the type of materials thatcan be used for drug delivery. In this invention, the sharpness andmechanical strength are separated from the drug delivery aspect of theimplant. The metals have the best combination of toughness and hardnessand thus are preferred in piercing the tissue (stainless steel metalused in the preferred embodiment shown in FIG. 15). The hollow cavity ofthe needle is used as a carrier of precast microimplant of desired shapeand size in the tissue as long as the shape chosen can easily pushed outof the cavity. The precast implant does not need sharp edge at distalend for tissue penetration. This approach enables the use of materialsthat are not mechanically strong but are useful for sustained drugdelivery or hydrogel soft materials that can encapsulate live cells andtherefore allows use of wider variety of materials. This also enables toeliminate the use of adhesive tapes and specialized applicator used topush the microneedle array inside the body. This method enables to useliquid drug carriers like vitamin E or sucrose acetate which cannot formconventional microarrays by themselves.

FIG. 22C shows a 3 by 3 microimplant array of fluorescent biodegradablecylindrical microimplants. The microimplants are inserted in the tissueto form microimplant array (2206). The microimplant array prepared asabove (2206) does not have sharp needle like edge but is implanted inthe tissue using AIA devices as described above. The implanted array canprovide sustained drug delivery or cells for therapeutic use. The array2206 was created by making the fluorescent cylindrical microimplants(size 100 mm diameter and 1000 mm height) by slicing a fluorescent 100micron diameter thread. The exemplary tissue is first treated withmicroneedle array or syringe needle to create array of artificial150-300 microns size cavities (cavity size must be larger than implantsize. The cylindrical implant is then inserted individually one by oneby hand or all at once using an array in array device as describedbefore. The array formed is imaged under blue light to visualize themicroimplant array. The inserted implants may further be immobilized byapplying tissue adhesive/sealant like DuraSeal or fibrin glue sealant oradhesive tape like band aid tape on top of the array to prevent itsmigration from the cavity.

In some embodiments, a unibody implant or combination of 1 to 10 unibodyimplants are preferred. The unibody implants have continuous solidstructure which enables to use them in AIA device. Unibody implants areeasy to push by the plunger array. If implant is not unibody type, forexample a loose powdered particles or drug encapsulated particles, itmay be difficult to push those using plunger array needles. Some of theparticles may reside in the device which may result into lower drug dosethan desired. However, the particles may be converted into unibodyimplant by sintering them or fusing them to form a sintered body whichhas uniform solid structure. The particles may also be bound using abinder additive and compacted under pressure to produce a unibody “greenbody mass” which can be pushed into body without leaving any residueinto the device. The particles may also be encapsulated in a hydrogelmatrix such as PEG based biodegradable hydrogels produced from PEG basedmacromonomers or PEG based crosslinkable precursors to form a unibodyimplant.

The array in array device described herein may be provided as a kitwherein microimplants and/or injectable compositions and AIA device maybe packaged together as a kit. The implants or injectable solutions areloaded in the AIA devices just prior to use.

In some cases, the implants delivered by the AIA device may undergochange in shape in contact with tissue or tissue fluids. For example,hydrogel based implant may increase in volume by absorbing water in thetissue and increase its volume by 1 percent to 10000 percent. In somecases, the device may shrink in size if the hydrogel is lyophilizedunder stretched conditions or under strain. Microimplants can bedesigned or made to change their shape after implantation. Change involume in one example for hydrogels. Biodegradable materials that canchange the shape are known in the art and such materials can also beused in preparing microimplants as discussed in this invention. Theadvantage of shape changed materials is that the implants cannot migrateor come out of skin tissue once implanted. The change in shape is not arequirement for this invention to work but can provide additionalbenefit if it is desired.

A comparison of conventional microneedle arrays made externally andmicroimplant array made using devices and methods described above isshown in Table 4.

TABLE 4 Comparison of conventional microneedle arrays made externallyand microimplant array made using devices and methods described above.Conventional microneedle Microneedles implanted using array used forImplantation devices and method described as above. The implantedmicroneedles The microimplant does not need must have sharp edge at havesharp edge. Can be used distal end for easy skin to implant cylindricaland insertion. This limits the other shaped materials. Enables shape ofthe implant. Cannot wider choice of implant shapes use cylindricalimplant with and materials. no sharp edge at distal end. Microneedlemust have certain No hardness and mechanical hardness and mechanicalstrength requirement for the strength for tissue microimplant.penetration. Limited to materials choices. Enables the use of wide rangeCannot be used with soft of materials. Can be used with hydrogelmaterials like soft materials and liquid gelatin hydrogel at ambientcarriers. temperature or liquid drug carriers like sucrose acetate.Difficult to use with live Can be used with live cells cells due toharness and to form live cell microarray solid state nature of theimplants. implant.Preparation of Prefabricated Porous Microimplants Xoated/Infused withDrug/Cells for Iimplantation in Microarray Dormat

This invention uses prefabricated microimplants, preferably unibodymicroimplants that can be implanted using AIA device or in artificiallycreated cavities. Some embodiments use prefabricated microimplants thatare porous in nature. The porosity may be filled with or porousmicroimplant surface may be coated with sustained drug releasecompositions or live cells for therapeutic use. The solid or polymersused in porous microimplant may also provide structural support to actas unibody implant which may help in pushing the implant via plungerarray needles as discussed before. The increased area created by theporosity, enables more surface area for the drug loading which enablesmore efficient utilization of microimplant surface. The total porosityof the implant may range from 5 to 95 percent, preferably 10-90 percentof the microimplant volume. The surface area of solid cylindricalmicroimplant can be substantially increased by creating artificialcavities or porosity. In one embodiment, several 0.005 cm diameter and0.01 cm dip cavities are drilled on the 0.1 cm diameter and 0.2 cmheight cylindrical microimplant. By creating 1 to 2000 cylindricalcavities (0.005 cm diameter and 0.01 cm height) on the surface,approximately 10 to 400 percent area (relative to the original area) ofthe implant can be increased. If a drug is released at 10 nanograms persquare cm area, then increased surface area would lead to substantiallyincreased drug concentration of the drug. The porous microimplant usedmay be biostable or biodegradable. The artificial porosity may becreated in many ways as known in the polymer foam preparation art. Theporosity preparation methods include but not limited to: creating a gassource inside the solid matrix such as carbon dioxide gas used to makebread porous via fermentation or by decomposition of sodium bicarbonate;removal of solvents by freeze drying or other methods; knitting orweaving of fibers; adding a porogen such as water soluble salt andleaching it out from the solid matrix and the like. For the purpose ofillustration, two exemplary methods of porous implant preparation havebeen provided. It is understood that other methods in the polymer foampreparation or tissue engineering scaffold preparation art, either knownor yet to be discovered, can also be used without limitation. In oneillustrative example, biodegradable poly (L-lactide-co-caprolactonecopolymer porous polymer is prepared by adding sodium chloride as aporogen. Briefly, poly(L-lactide-co-caprolactone copolymer is dissolvedin organic solvent such as tetrahydrofuran or dioxane. Dioxane ispreferred because it can be removed by freeze-drying process. Thedioxane solution of the polymer is mixed with sodium chloride crystalsto make a suspension. The sodium chloride crystals are ground by mortarand pastel or any other means and sieved to obtain a desired particlesize and distribution fraction. The polymer and salt suspension isstirred to mix the crystals uniformly and frozen quickly using liquidnitrogen to form a frozen solid matrix. The frozen solid is subjected tolyophilization or freeze-drying process to remove the solvent bysublimation process. The freeze-drying is conducted at temperaturearound −10 to zero-degree C., typically around −5 degree C. The driedsolid is exposed to water to dissolve out the sodium chloride in thewater and thus forming a solid polymer structure with empty spacevacated by leaching out sodium chloride and solvent dioxane. By changingthe amount of solvent, sodium chloride and its particle size and shape,a wide variety of porous implant structures with varying degree ofporosity can be created. For example, salt may be added up to 95 to 5percent, preferably 90 to 10 percent and even more preferably of the 70to 20 percent of the volume of microimplant. Many water soluble ororganic solvent soluble porogen or leaching agents may be used and theseinclude inorganic or organic salts, prefabricated microspheres withuniform particles size, organic and macromolecular substances likepolyethylene glycol and the like. The preferred materials includematerials that have well defined particle size and distribution whichmay include but not limited to: commonly used salts and compounds suchas sodium chloride, potassium chloride, sugar, glucose, and the like. Inchoosing the porogen, it is essential that it can be extracted withoutaffecting the polymer in which it is incorporated. For example,extraction solvent must be a non-solvent for the polymer being used. Inone embodiment, poly(L-lactide-co-caprolactone copolymer is used whichis water insoluble and therefore water extractable porogen such assodium chloride may be suitable to make the porosity. For hydrophilic orwater soluble polymers like collagen or gelatin or poly(vinyl alcohol),polyvinyl pyrrolidinone, hydroxy ethyl cellulose, hydroxy propylcellulose and the like sodium chloride may not be preferred because itcan also dissolve/swell the water soluble polymer. Organic solvents suchas methanol acetone, which are non-solvent for collagen or gelatin, maybe used for porogen extraction. In one exemplary embodiment, methanolsoluble microspheres are used as a porogen and are removed from methanolextractions. The list of solvents and non-solvent for many polymers canbe found in Polymer Handbook. For water soluble polymers andmacromolecules such as collagen or gelatin, the water itself may be usedto create porosity. The free water in the aqueous polymer solution canbe removed by lyophilization to create porosity in the polymer. Inanother illustrative example, ethyl methacrylate microspheres, which canbe removed by methanol extraction, have been used. The microspherescreate spherical voids in the polymer solid. Solvent may be added from0.1 to 60 percent relative to polymer weight depending on the solventused. Generally polymer is dissolved 1 to 50 percent relative to solventweight, preferably 5 to 30 percent. Many types, beads/microspheres thatmay be useful are commercially available from suppliers likePolysciences, USA. Porous structure may be created in many biostable andbiodegradable polymers as defined previously. The polymers may behydrophobic or hydrophilic. The polymers may be crosslinked orthermoplastic. In general polymers must be biocompatible and suitablefor implantation in the human or animal body. Among biodegradablepolymers, synthetic or natural polymer or macromolecules as discussed inearlier sections may be used. The preferred biostable polymers includebut not limited to aliphatic and aromatic polyurethanes, polycarbonatepolyurethanes, polyether polyurethanes, silicone polyurethane blockcopolymers, silicone rubbers, polydimethyl siloxane copolymers,polytetrafluoroethylene, expanded polytetrafluoroethylene, polyethylene,polypropylene, polyamide, polyamide block copolymers and the like. Inanother exemplary embodiments, methanol or water extractable PEG is usedas a porogen to make a porous structure. In one exemplary embodiment, anatural hydrophilic natural polymer is used to create porosity via PEGextraction. A collagen solution is frozen with poly(ethyl methacrylate)beads as a porogen and lyophilized. The freeze-dried collagen-beadcomposite is then subjected to methanol extractions to remove themicrospheres and create porosity in the collagen. The collagen porousimplant may be crosslinked with1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or glutaraldehydeor PEG based water soluble crosslinkers to improve its degradation timein the body. Treating the porous collagen porous implant with 0.1 M EDCsolution in 50 percent acetone for 8 hours can form crosslinks in thecollagen. By changing EDC concentrations and reaction time, collagendegradation time in the body can be changed. EDC crosslinked porouscollagen also serves as exemplary crosslinked hydrophilic polymer basedporous implants suitable to be used as drug carriers. Other carbodiimidebased crosslinkers may also be used.

Loading of Drug/Cell Comprising Compositions in the Porosity ofPrefabricated Porous Microimplant to Form Microarray:

Once the porous structure is obtained, the porous structure can bepartially, substantially or completely filled or coated with drugreleasing compositions, which may comprise, a drug or drug with carrieror live cells. The list of drugs that can be used is provided in thedefinition section. The carrier may be non-polymer, ceramics, minerals,polymers or macromolecules that may be suitable for use in the human oranimal body. The carrier may be liquid, solid, waxy, gels, semisolids,or viscous liquids. The preferred carrier may be biostable andbiodegradable polymers. The polymers may be hydrophobic or hydrophilic.The polymers may be crosslinked, non-crosslinked or thermoplastic. Ingeneral polymers must be biocompatible and suitable for implantation inthe human or animal body. Among biodegradable polymers, synthetic ornatural polymers/macromolecules may be used. The biodegradable polymersmay be hydrophobic or hydrophilic. The biodegradable polymers may becrosslinked or non-crosslinked. The crosslinking may be done viacondensation polymerization or via free radical polymerization. Thepolymers may be random or block or graft copolymers. The polymers may belinear, graft or branched. The hydrophobic polymers include, but are notlimited to, polymers, dendramers, copolymers or oligomers of glycolide,dl-lactide, d-lactide, l-lactide, caprolactone, dioxanone andtrimethylene carbonate; degradable polyurethanes, polyamides,polyesters, polypeptides, polyhydroxyacids, polylactic acid,polyglycolic acid, polyanhydrides, and polylactones. Hydrophobicpolymers also include polyhydroxyalkanoates which are polyestersproduced by microorganisms such as poly-(3-hydroxybutyrate),3-hydroxyvalerate, 4-hydroxybutarate, 3-hydroxyhexanoate,3-hydroxyoctanoate. Preferred hydrophilic polymers include, but are notlimited to, polyethylene glycol-polyhydroxy acid pr polyethyleneglycol-polylactone copolymers (PEG-PL copolymers), polyvinyl alcoholco-polylactone copolymers, and derivatives of cellulose, collagen,gelatin, albumin, fibrinogen, keratin, starch, hyaluronic acid anddextran. The PEG-PL copolymers are most preferred. PEG-PL copolymerssuch polyethylene glycol-polylactone copolymers can have a range ofproperties from hydrophobic to hydrophilic depending on the amount ofPEG incorporation in the copolymer and molecular weight of PEG andpolylactone. In one exemplary embodiment, drug solution is infused inthe body of a porous implant without the use of carrier polymer. Thedrug loading can be 1 to 40 percent relative to the weight of thepolymer or the drug can occupy 1 to 90 percent of the pore volume. Inanother exemplary illustration, a synthetic biodegradable polymer isused as drug carrier for the drug. The drug and biostable polymer orbiodegradable polymer is dissolved in a common solvent and the solutionof the polymer is exposed to the porous implant. The solvent for thepolymer and drug infusion should be a non-solvent for the porousimplant. In the illustrative example, 10 ml tetrahydrofuran or1,4-dioxane, 900 mg of poly (PLGA, lactide-co-glycolide)(lactide:glycolide (50:50), molecular weight 30000 to 60000 g/mole.) and100 mg (approx. 10 percent loading relative to weight of polymer)Latanoprost are mixed until homogeneous solution. A porous collagenimplant or commercial collagen microimplant from Odyssey Medical, Inc.(size 0.4 mm diameter×1 mm length) is used as a substrate. 25 drycollagen porous microimplants are incubated in the PLGA and drugsolution in organic solvent such as dioxane or THF and after completepenetration of the solution into the pores, the excess solution isremoved and the implants are added in 10 ml PBS solution in glass vialto precipitate the polymer in the pores of the implant.

After incubating for 6 h, (PBS solution is changed every hour), theimplants are lyophilized. Alternatively, after complete penetration ofdioxane-polymer-drug solution in the pores, the implant is taken out,wiped to remove excess solution from the surface and frozen immediatelyusing liquid nitrogen and lyophilized to remove the solvent. The poresin the microimplant now has biodegradable polymer with drug. Thedrug-polymer is either precipitated in the pore or freeze-dried in thepores. If the pore volume is high, then large amount of volume isoccupied by the drug releasing composition. The implants are loaded anddeployed in the array format using AIA device as described before. Thedrug is released in a sustained manner due to diffusion and/orbioerosion/biodegradation of the biodegradable polymer. The release ratewill depend on the biodegradation rate of the polymer used, drugsolubility in the polymer, drug loading in the polymer (2 to 60 percent,preferably 5 to 40 percent relative to weight of the biodegradablepolymer). Since dioxane is a non-solvent for the porous implant, theporous implant can maintain its mechanical integrity which is helpful inpushing the implant in the tissue using AIA device. In some embodiments,a biocompatible liquid carrier may be infused in the porosity ofmicroimplants. The liquid carriers that may be used include but notlimited to: vitamin E or its derivatives, vitamin E acetate liquidpolylactone or polyhydroxy polymers or copolymers or copolymers,PEG-polylactone copolymers, PEO-PPO-PEO polylactone copolymers, sucroseacetate, natural or synthetic oils and fats, fatty acids, fattyalcohols, oleic acid and its derivatives and the like. The porousimplant provides a matrix and unibody properties for the microimplantand liquid carrier and drug infused/coated in the porous structureprovides sustained release of drug. In another exemplary embodiment, thedrug compositions may be injected directly into pores or artificialcavities created in the implant body.

The unibody microimplant array formed is preferred to be colored orfluorescent or combination thereof. The color or fluorescent enables tosee the implanted array after it has been formed or implanted in thetissue. Color may also help to encode/decode certain information such asmanufacturing batch number, expiry date of the product and the like.Colored compounds could be added in many ways. Color could be covalentlybound, ionically bound, stained or physically mixed with themicroimplant composition. Preferably, the colored compound isbiocompatible and biodegradable. Even more preferably the coloredcompound used is selected from the compounds which have history of usein human or history of use in medical devices or pharmaceuticalcompositions. The percent of colored or fluorescent compound could beadded in microimplant may range from 0.05 percent to 10 percent,preferably 0.1 to 5 percent relative to the weight of microimplant.Color also could be added as colored biodegradable microparticles ormicrospheres. In some cases, the color or fluorescent compound is addedto the microimplant or hydrogel matrix used in the microimplant array.In one illustrative embodiment, fluorscein is covalently attached tocollagen or bioprosthesis tissue. This was achieved by treating/reactingthe fluorscein and collagen/bioprosthesis tissue in presence ofN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) as acatalyst at ambient temperature in aqueous buffered modicum around pH 6to 7. The fluorscein linked to the collagen chains via ester or amidebond. The fluorscein bound collagen or tissue shows green fluorescencewhen exposed to blue light. Color or fluorescent compound can also becovalently bonded to synthetic hydrogel biostable or biodegradablematrix used in microimplant array formation. Preferably the synthetichydrogel matrix is a biodegradable hydrogel comprising PEG or PEG basedcrosslinked hydrogel.

Implantable Biodegradable Metal Based Arrays for Drug Delivery

This invention discloses microneedle based drug delivery arrays derivedfrom biodegradable metal. Since metals offer the best combination ofmechanical properties for easy skin penetration, we have discovered thatmicroneedles made from biodegradable metals could be useful in drug/celldelivery application. This invention discloses biodegradable microneedlearrays made using biodegradable metal. The inventive arrays havemicroneedles with sharp edge. The microneedles are made usingbiodegradable metal. The array also has a backing layer wherein needlestemporarily are attached to the backing layer via adhesive layer,preferably a pressure sensitive adhesive layer. The purpose of thebacking layer is to hold the array needles in place temporary untilimplantation and detach itself easily once array needles are implantedin the skin. The adhesive layer is attached to a flexible polymer film,leather or woven/knitted textile fibers or rubber like materials. Theneedles are attached perpendicular to the backing layer and the sharpedge is pointing away from the backing layer surface. Preferably theneedles are attached in a matrix array format. The microneedles attachedto the adhesive backing layer make the implantable drug delivery devicearray.

FIG. 26 shows partial schematic representation of various configurationsof biodegradable metal based, preferably magnesium alloy based,microneedles that can be useful in making implantable drug deliveryarrays. FIG. 26A schematically shows a biodegradable magnesium alloybased hollow array needle (2601) with sharp distal edge (2602) for easytissue penetration. The hollow cavity of needle is partially orcompletely filled with sustained drug delivery composition such as PLGApolymer with a drug (2603). FIG. 26B shows a schematic of anillustrative array needle 2604 whose external surface is partially orcompletely coated with biodegradable sustained drug delivery composition(2605). The composition 2605 may have one or more coating layers and oneof them may be a rate controlling layer without a drug. FIG. 26C shows ahybrid needle wherein the tip of the needle (2606) is made up usingbiodegradable metal for easy skin penetration and the drug deliveryportion (2607) is made biodegradable polymer/hydrogel with sustaineddrug/cell delivery composition. FIG. 26D shows schematics ofillustrative needles with artificially created porosity for drugdelivery. The illustrative configurations create artificial porosity toincrease surface area and to create a space for drug deliverycompositions. FIG. D1 shows a biodegradable metal array needle (2608)with distal sharp edge wherein wedge shaped micro pockets are createdinside the needle surface and then filled with drug delivery composition(D1, 2609). In another variation (D2), rectangular portions have beencut out in the needle body to create a space for drug deliverycomposition filling material (2610). In another variation (D3), severalartificial microcavities of various shapes (cylindrical in thisillustrative case) or holes are created by the needle surface/body andthe cavities/holes are then filled with injectable drug deliverycompositions (PLGA with rifampin as an example, 2611). FIG. 26E showsillustrative microneedle array device with biodegradable metal basedmicroneedles attached to a flexible backing material. Four microneedleswith holes/cavities filled with biodegradable drug delivery compositions(2611, D3) are attached using a pressure sensitive adhesive (2613) to aflexible backing material (2614) in an implantable array format tocreate an exemplary biodegradable microneedle array device. The needlebase is attached to the adhesive and free distal end with sharp edge isused for tissue penetration. The device is inserted in the skin tissueby pressing the backing layer with needles sharp ends facing the skinand implanted in the skin. The backing/adhesive material is removedleaving behind the array in the skin. The metal array and drug deliverycompositions are biodegradable and provide a drug for local or systemictherapeutic effect.

In one illustrative and non-limiting embodiment, a 100 mm length, 100 mmwidth and 500 micron thick magnesium alloy (AZ31, Magnesium 96 percent,Aluminum 3 percent and Zinc one percent) foil from GoodfellowCorporation, Coraopolis, Pa., USA (product code 343-198-08) is used tomake an implantable array (Example 25). In another embodiment, a hollowmicroneedle based needle is used to prepare an array device (Example25). The hollow cavity of the microneedle is filled with sustained drugdelivery compositions. Yet in another embodiment, a magnesium alloybased microcylinder is coated with drug delivery composition withmultiple layers and the microcylinder is implanted using AIA devicedescribed in this invention.

In all the cases, a biodegradable metal is used in variety of differentdesigns and configurations to make the implantable array based drugdelivery device (Seitz J et al., Adv. Healthcare Mater., volume 4, page1915-1936, 2015). The biodegradable metal used could be any metal thatdegrades safely in the body in a period from few days to few years,preferably in 7 days to 270 days. Many alloys of calcium, magnesium,aluminium, zinc, manganese, iron and other metals are known to degradesafely upon implantation. Preferred metals do not create extreme basicor acid local environments upon implantation and is safely removed bythe body via biodegradation process such as hydrolysis or othercorrosion type mechanism. Metal alloys based on magnesium are preferreddue to its long history of use. The metal only serves as a structuralpart in the inventive device. The drug delivery function is done using abiodegradable polymer or hydrogels in the device. The biodegradablepolymers and hydrogels along with drug/s can be coated on the needlesurface or may be infused in the hollow cavities of the microneedles.Hydrogel in dry form or dehydrated may also be used. The drug deliverycompositions may be infused in the grooves, wedges, or holes created inthe microneedle surface or the body. In one exemplarity embodiment, ahollow microneedle is used to create a microneedle array and the hollowcavity is filled with biodegradable polymer composition with rifampin asmodel drug. In another embodiment, the surface of the microneedle arrayis coated with one or multiple layers of biodegradable polymers and thecoatings are used for sustained drug delivery. Some layers in thecoating are used to control the rate of release of the drug. In anotherillustrative embodiment, several holes are created in the microneedlearray body and the holes are used to load the drug deliverycompositions. The holes could be filled in two or more layers of drugdelivery compositions. The embodiment uses cylindrical holes or groovesbut any shape can be used.

Cylindrical, triangular, square, rectangular, pentagonal, hexagonal orirregular shaped holes or grooves may be used. Cylindrical holes/groovesare most preferred.

In some embodiments, porous magnesium alloy based arrays may be preparedby sintering alloy powders and the porosity is then filled withinjectable composition comprising drugs/cells. Porosity may also becreated using methods such as laser drilled holes as discussed before.Compositions comprising drugs/cells may be infused in the porosity ormay be coated on the needle surface as lyophilized powders or solids orliquids or semisolids or as injectable compositions discussed in thisinvention.

The physical size and dimensions of microneedles in the exemplaryembodiments is for illustration only. Those skilled in the art canrecognize that microneedles with variety of sizes, shapes, heights,volumes, number of needles per array, total area of the array and thelike can be made to obtain a microneedle array of desired size andshape. The average diameter of microneedle in the array may range from 1micron to 3500 microns, even more preferably 5 microns to 2600 microns.The height of the needle in the array may range from 5 microns to 5000microns, preferably 10 microns to 3000 microns, even more preferably 10microns to 2000 microns. The shape of array needle may range fromstraight obelisk, negative-beveled obelisk, cylindrical, pyramidal,conical, trigonal, tetragonal, pentagonal, hexagonal, pyramidal,irregular and the like or combinations thereof. The distance betweeneach needle in the array may range from 1 micron to 10000 microns,preferably 3 microns to 5000 microns, even more preferably 5 microns to2000 microns. The volume of each needle may range from 1×10E-12 to 0.05ml, preferably 1×10E-10 to 0.03 ml, even more preferably 1×10E-10 to0.01 ml. Total number of needles may be greater than 4 per squarecentimeters or may range from 4 to 500 per square centimeter. Totalnumber of array needles may be greater than 4 or may range from 4 to6000, preferably 4 to 2600, most preferably 4 to 1000 per array. Thearray has a backing attached to the base which provides an area wheremechanical force or pressure is applied so that all needles of the arraycan be inserted in the tissue at the same time. Variety of basematerials for backing materials known in the art can be used. Theseinclude but not limited to: nylon, cotton, different woven textilematerials, metals and ceramic and the like. The microneedle array needlewith backing material is a one of the several preferred designs. Devicessuch as “array in array” or AIA devices, described in this invention,may also be used to infuse biodegradable metal based microimplants inthe skin. The microneedles may be loaded in hollow cavity of the basearray needle and injected in the skin tissue either individually, or onerow at a time or one column at a time or whole array at once. A specialapplicator may be designed for each type of insertion and used toimplant biodegradable metal based arrays disclosed in this invention.

Applications of Methods and Compositions Proposed in this Invention

Following are some of the clinical applications where the methods andcompositions described in this invention may be useful. The proposedapplications do not in any way limit this invention to other medicalapplications. Many embodiments use skin tissue as a main tissue. Theadvantage of skin tissue is that it is external and with large surfacearea. In some cases, if microimplants need to be removed due to allergicreaction to the drug or some other medical factors, the removal may bepossible for skin inserted microimplants. Such a removal is not possiblewith conventional injection of sustained drug delivery biodegradablemicrospheres in intramuscular tissue. The medication or implantmaterials may be removed using methods such as laser treatment similarto used in tattoo removal procedures. The laser induced heat treatmentcan also destroy and/or denature and/or eliminate biological activity ofthe drug. The implant may also be removed by surgical procedure ifpresent in the skin surface.

Encoding Information in the Array

In one illustrative embodiment, one cylindrical microimplant withradio-opaque properties, one cylindrical implant with fluorescentproperties and 7 PLGA microimplants with moxifloxacin are obtained.Fluorescent and radio-opaque microimplant may not have drug but aredesigned to help to identify and tag the array after implantation. Theimplants are loaded in the array form using AIA apparatus as describedin this invention (3 by 3 array). During loading, radio-opaque implantis loaded at the center and fluorescent microimplant is located at oneof the four corners. The positions of the radio-opaque and fluorescentmicroimplants can be used to code and decode certain information, liketype of surgery used, gender of the patient and the like. Thus, the useof array format and its location in the array could be used to convey orrelay certain information which may be relevant to the doctoradministering the treatment, government regulating agency, patient andthe like. The use of visualization agent and its location in the arraycan be used to convey or relay that information. Variables like positionor location in the array, fluorescence wavelength (red verses greenfluorescence), radio-opacity, ultrasonic imaging visibility, MRI imagingvisibility and the like can be used to code and decode certain usefulinformation in the array. The embodiment described above is forillustration only. Those skilled in the art can recognize that manyvariations are possible such as number of microimplants in the array,geometry of array (circular versus rectangular array as an example),shape of microimplant and the like can be used to relay usefulinformation that can be used to improve medical treatment and/or itsservice and regulatory component. Some of the spaces or positions in themicroimplant array may be used to load certain sensors such astemperature sensor, pressure sensor, insulin sensor, radio frequencyidentification tags (RFID), cell viability indicators, pH sensors,chemical sensors, biological entity sensor, bacterial activity sensor,viral activity sensor and the like. Generally greater than 10 percent,preferably greater than 50 percent and preferably greater than 95percent positions in the in the microimplants array described in thisinvention may be used for implants with drugs or cells. Remaining otherpositions may be used to load microimplants with visualization agents orsensors and can be used in coding and decoding certain information.

Nail and Bone Infections

It is generally well known that bone infections are difficult to manage.The infected area in the bone and nail may be first accessed, a porosityis created in the affected area and surrounding area and the antibioticcompositions such as PLGA microparticles with antibiotic or PLGAsolutions with antibiotic may be used to fill the porosity. Anti-fungaldrugs may be given via cavity filling process to manage fungal nailinfections.

Pain Management

Local anesthetic agents such as bupivacaine can potentially be used inmanaging local pain. A pain associated with surgical wound may bemanaged by directly forming a microarray based implants in areas oflocal pain or surgical wound site. Local anesthetic such as bupivacaineis released via in situ formed biodegradable microarray for 1 to 5 daysuntil surgical wound is healed.

Cardiovascular Applications

The damaged heart tissue is extremely difficult to regenerate. Stemcells therapies can help but distribution of cells in affected areas isdifficult to manage. The cells delivery via in situ microimplantformation may be useful as described in this invention. Severalmicroimplants containing stem cells which can be converted into heartmuscle cells may be implanted via in situ generated microimplants asdisclosed in this invention or preformed implants with cells. The sizeof implant, number of implants, and depth of implants may be tailoredfor a specific need of a patient. The damaged heart tissue may beexposed to laser based method to create micro-porosity and the holes maybe filled with stem cells and encapsulated in fibrin glue matrix orother suitable biodegradable hydrogel matrix. The encapsulated stemcells reproduce in the cavity and produce new heart muscle which mayhelp in improving cardiovascular function of the heart. Alternatively,cell containing microimplants can be made just prior to surgery andimplanted using devices described in this invention. Also, microneedlearrays containing frozen stem cells may be implanted in treated areaunder frozen conditions (cryopreserved cells) as described in thisinvention. Many methods and compositions for delivering stem cells aredescribed in this invention, ultimate choice will be determined by theend user and best desirable clinical outcome. The cell suspension in PBSor tissue culture medium may also be infused into the heart tissue usingoscillating needle apparatus as described in this invention. This waylarge area of the organ can be infused in a relatively short period oftime and with uniform distribution of therapeutic cells. Theexperimental conations used must provide live cells after implantation.

Therapeutic arrays or coated biodegradable threads reported in thisinvention may be used to deliver anti-restenosis drug like paclitaxel,everolimus, sirolimus which may be delivered at the vascular graftanastomosis sites during vascular graft implantation procedures orcoronary bypass surgery procedure.

Ophthalmic Drug Delivery

The inventive compositions can be useful in many ophthalmic indicationsincluding but not limited to reducing inflammation and pain aftercataract and other ophthalmic surgery, controlling viral and bacterialinfections, age-related macular degeneration and the like. In oneillustrative embodiment, a drug and polymer solution is applied onscleral tissue and tissue is perforated using a microneedle array. Thesolution is deposited in artificial cavities created by the array. Thedeposited solution precipitates in the scleral tissue entrapping thedrug which is then released in a sustained manner. In anotherembodiment, a hollow microneedle array such as 33 MP array is used todeposit a polymer and drug solution or suspension containingbiodegradable microspheres containing dexamethasone as exemplary drug.The deposited drug is released in to eye in a sustained manner. Inanother embodiment, preformed cylindrical PLGA microimplants containingtotal of 0.4 mg dexamethasone are deposited in 5 by 5 array format inscleral tissue using AIA device described in this invention.

The implanted array releases the dexamethasone in a sustained mannerover period of 14-30 days. Preferably the array is colored orfluorescent for easy visualization and monitoring.

Diabetes Management

Pig islets are available in abundant supply and insulin produced by pigislets is well tolerated by the humans. Microimplant array containingapproximately 1 million pig islets are believed to be sufficient to actas artificial pancreas for humans. The pig islets can be implanted underthe skin with or without immunoisolation. Depending on the density ofarray, 1 million pig islets can be implanted using up to 15×15 cm areaof the skin. Example 15 and 16 provide several illustrative embodimentsthat can be used to implant islets under the skin. It is preferred animmunoprotective matrix is used in the array for islet encapsulation andthe encapsulation matrix forms a minimum amount of scar tissue. The scartissue can potentially prevent insulin diffusion from the islets and mayalso limit the nutrient supply which is necessary for survival of cells.PEG based hydrogel surfaces are known to reduce scar tissue formationand are therefore are preferred in this application. The implantedislets can continuously monitor glucose concentration in the tissuefluid and release insulin necessary to control the glucose in the tissuefluid. This way, better control over glucose managements is possible.Depending on the immunoprotection provided, the patients may need totake immune suppressing drugs to protect islets from immune system.Since this implantation is done in the skin tissue, adverse reactiondeveloped by animal cells may be reduced/eliminated by destroying ordenaturing the cells in the skin implant using laser based methods orsurgical based methods as discussed before. Alternatively, drugs such asinsulin and other drugs that control level of sugar in the blood may beused to form microimplant arrays as described in this invention. Suchdrugs may be encapsulated in biodegradable polymers and released in asustained manner for better control of sugar in the blood.

Management of Iron Deficiency

Iron deficiency or anemia associated with lack of iron in the blood isone of the most important health issues in the world today, especially,in the third world countries. Iron deficiency is caused by many factorswhich include mensural cycle in females, GI bleeding, malnutrition,premature birth, parasitic infection and the like. Iron deficiencyaffects cognitive development of children from infancy through toadolescence. Iron deficiency is believed to be associated with increasedmorbidity rates. Certain oral medications have also tendency to reduceiron content in the blood. Iron deficiency is generally managed throughoral supplements and is not considered to be very reliable method tomanage anemia. Oral therapy has lower bioavailability of iron and hasside effects such as constipation. Oral therapy also has complianceissue because patients may not complete the prescribed oral dose. Severeiron deficiency can be managed via intravenous route but requirescareful monitoring in hospital settings and has certain risks such asanaphylaxis, and cardiovascular complications. U.S. Pat. No. 8,821,945reports iron complexes delivery using iontophoretic method.Iontophoretic method uses electric current to drive iron complex acrossskin barrier. The use of electric current may have potential to causeburn and other types of injury. Iron can be delivered using compositionsand methods described in this invention. The iron complex, such asferric pyrophosphate is transported across the skin barrier anddelivered in the skin tissue. Once transported, the complex is believedto be absorbed by the surrounding tissue and used in reducing irondeficiency by variety of known and unknown biological mechanisms. In oneillustrative embodiment, aqueous ferric pyrophosphate or iron sucrosecomplex solution is injected in the skin tissue using an oscillatingneedle and using hollow microneedle array. An image of treated tissuewith iron sucrose complex is shown in FIG. 9A. The treated tissue hasdark brown color due possible tattooing effect of the drug. Thetattooing effect is believed to be caused by the insoluble oxidationproducts caused by reaction of free iron ions with tissue proteins. Theinfusion of ferric pyrophosphate generally did not cause significanttattoo effect indicating the iron remain complexed with pyrophosphateion and does not provide free iron for tattooing effect. In anotherembodiment, the ferric pyrophosphate solid powder is ground, sieved toprovide 300 microns or less particle size. The sieved powder issuspended/dissolved in glycerol and tattooed using oscillating needle.The solid powder has higher density than the solution and thus may helpto reduce the amount of materials that needs to be delivered across theskin tissue. In another embodiment, a PLGA solution in NMP is mixed withsieved iron pyrophosphate powder. The suspension is injected usingtattoo needle in the skin tissue. The NMP is dissipated in the tissue,entrapping iron pyrophosphate in the PLGA polymer which is released in asustained manner. A release profile of iron released from theprecipitated polymer is shown in FIG. 20. As seen from the figure, ironis released in a sustained manner over a 4 day period. The encapsulationof iron is believed to reduce the interaction of iron from the skintissue proteins and hence may further help to reduce tattooing effect.The illustrative embodiments use two iron compounds ferric pyrophosphateor iron sucrose complex, but the use of iron pyrophosphate is preferreddue to its stability and its ability to convert into useful iron complexthat can be used in reducing iron deficiency. The iron complex could beeither ferrous or ferric salts of iron. Iron complexes that can be usedand may include but not limited to: ferric carboxy maltose, ferrictrisglycinate, ferrous gluconate, iron-dextran complex, iron-dextrin,ethylenediamineedetate iron complex, ferric ammonium citrate,ethylenediaminesuccinate iron complex, ferric citrate, ferric manganesecitrate, iron-sorbitol-citric acid complex, ferrous fumarate, ferricgluconate complex, glycol ether diaminetetraaceticacid iron complex andthe like. Additional list of ferrous or ferric complexes can be found inU.S. Pat. No. 8,821,945, cited herein for reference only. The preferredamount of iron that can be infused in the tissue may range from 5 to 100mg, preferably 10 to 50 mg, even more preferably 10-30 mg. The dose ofiron will depend on the patient factors such as weight and deficiency ofiron and the like.

Iron also can be delivered using hydrogel based dissolvable array. Inone illustrative embodiment, a sodium hyaluronate based dissolvable withferric pyrophosphate array was made (FIGS. 9B1 and 9B2). The array caneasily penetrate in skin tissue and dissolves in the skin tissue under10 minutes. The dissolved array delivers ferric pyrophosphate in thetissue. The dose of the iron can be managed by iron concentration ineach needle and number of needles/arrays inserted in the tissue.

Arrays Containing Botulinum Toxin and Other Protein Drugs/Vaccines

Drug Delivery without Making Drug Solution.

Delivering Drug in a Solid State and in the Unibody Microimplant Form.

Botox® is a trade name for Botulinum toxin. Botox is neurotoxic proteinproduced by the bacterium Clostridium and is sold to treat variety ofmedical conditions. Biodegradable arrays can be made comprisingBotulinum toxin and delivered in the solid state into skin tissuewithout the use of dilution and injection. The use of Botox array can bemade for specific medical application with each array containing 0.1 to30, preferably 0.5 to 20 units of Botulinum toxin. Preferably sucharrays are colored or fluorescent so that their implantation can bemonitored and/or documented. The distribution of the drug can beachieved over a large surface areas under the arm pit for excessivesweat management or on the face for wrinkle management. The applicationof Botulinum toxin array can be relatively pain free for the patient.The recommended shelf life of Botox solution is 48 hours and this canpotentially lead to waste of expensive drug if not used immediately.Since this delivery of method does not involve solution preparation, thewastage of Botox during application can be potentially eliminated. It ispreferred that each Botox microimplant is present as a unibody which canbe pushed using the methods and devices described in this invention.Vaccines may be delivered using the same manner as Botulinum toxin asdiscussed before.

Botox is generally sold as a dry lyophilized protein powder with eachvial containing 50 or 100 units dose of the drug. The Botulinum toxinconcentration is generally present at around 1 nanogram level which ishard to detect and manage in solid state. In this invention, a bulkingagent is added to increase the mass of the total solids present in thedevice. In one illustrative embodiment, the Botox vial containing 100units is diluted with sterile 0.1 ml of 0.1 percent sodium hyaluronate(mechanical property enhancer and bulking agent) or human serum albumin(10 percent solution) in PBS buffer (pH 7.4) and 0.1 mg sodiumfluorscein (as a coloring/fluorescent agent) or F and D and C bluenumber 2 as a colorant. The 3.53 microliter of the Botox solution isloaded 100 cavities of base array of 10 by 10 AIA apparatus as discussedin Example 22. The apparatus has 100 cylindrical cavities with 500micron height and 310 micron diameter cavities. The filled solution isfrozen and lyophilized in the cavities. A 10 by 10 plunger array of theAIA apparatus is applied on top of the base array aligning its needlebut not inserted. A polyethylene 2 mm spacer lock is kept between thebottom and top array to prevent accidental insertion of plunger array inthe base array. Each 100 microneedle array contains approximately 3.53units of Botulinum toxin (0.0353 units per microimplant, total 100microimplants). In case of Botox, most of the solid in the implant,preferably over 80 percent, preferably greater than 50 percent of theimplant volume is occupied by the bulking agent such as hyaluronic acidor human serum albumin or compounds like sugars and polymers used inmaking dissolvable array. Generally, the bulking agent and otherpharmaceutical additives such as colorant, antioxidant, stabilize,lyophilization agent and the like may occupy up to 1 to 99 percent oftotal implant volume, preferably 5 to 95 percent of the total implantvolume. The amount of Botulinum toxin per implant is generally higherthan 0.01 units per microimplant and may range from 0.01 units to 30units per implant, preferably 0.01 to 20 units per implant and even morepreferably 0.01 to 10 units per implant. The device is terminallysterilized and used on the skin tissue. During the usage, the base arrayneedles are inserted in the tissue, spacer is removed and the plungerarray is pushed to inject the fluorescent implant in the tissue. Onceinjected, both the arrays are taken out leaving behind the implant inthe tissue. Under blue light, implantation of the injected microimplantarray is visible as green array. Multiple arrays may be used to treatmore areas on the skin if needed. The sodium hyaluronate and fluorsceindissolve away leaving behind Botulinum toxin for therapeutic effect.This method delivers Botulinum toxin in the solid state without dilutionin saline as used in current practice. If Botulinum toxin containingarrays are delivered using AIA device/apparatus as discussed in thisinvention, the microimplants with Botulinum toxin can be pushed out inthe skin tissue one implant at a time from the base array, or onerow/column at a time or all implants can be pushed at once.Alternatively, implants can be delivered at a predetermined sequence ifthe plunger array movement is controlled electronically by computeralgorithm. The ultimate choice will determine the dose of Botulinumtoxin per implant and total dose needed per treatment site. In somecosmetic applications, the fluorescence and color of the implant is onlyvisible under blue light and not under normal condition. This way thepresence of the implant does not alter cosmetic appearance of thesubject. This is achieved by many methods but in one illustrativeexample, minimal amount of fluorscein is used and due to highsolubility, the dye disperses quickly in the tissue. In someembodiments, the preformed solid microimplants may be impregnated usinga sterile Botox solution of desired concentration and then impregnatedimplant in solid state is then inserted in the skin tissue as describedbefore. However, microimplants that are infused/encapsulated with Botoxdrug in a manufacturing setting are preferred because solutionpreparation step is not required during implantation step.

Other protein/biologics drugs such as vaccines (Influenza vaccine as anexample) can be delivered in the solid state using similarly to Botox asdescribed before. Many biologics drugs such as Etanercept (Enbrel®) orAdalimumab (Humira®) are injected as solutions to treat rheumatoidarthritis and such drugs also may be formulated into unibodymicroimplants in solid state and then implanted in an array format asdescribed in this invention.

Adrenalin or Epinephrine or Adrenaline is a drug used to treat severelife threatening allergic reaction approved (anaphylaxis). Epinephrineauto-injectors such as EpiPen® is used by a person with a history of asevere allergic reaction. Adrenalin could be formulated intomicroimplants that can be implanted in microarray format as described inthis invention to provide a total dose of 0.3 or 0.15 mg per array totreat severe life threatening allergic reaction.

In an embodiment, a drug delivery microneedle array device comprises twomicroneedle arrays; a) i) first array comprises one base plate with topand bottom surface and x number of hollow microneedles with internaldiameter d protruding from bottom base surface wherein x is grater than3; ii) the hollow microneedles have sharp cutting edge at distal end andproximal end opening on top surface; b) i) second array comprises onebase plate with top and bottom surface and x number of solidmicroneedles with diameter 5 percent or more smaller than d andprotruding from bottom base surface wherein x is greater than 3; ii) thesolid microneedles have non-cutting surface at distal end; iv) thematrix arrangement of needles is identical to first array needles; c)the second array needles can be inserted in hollow cavity of first arrayvia needle opening on top surface of first array after aligning centerof needles of both the arrays.

In an embodiment, a drug delivery microneedle array device comprises twomicroneedle arrays and one cartridge for holding of microimplants withdrug/cells; a) i) first array comprises one base plate with top andbottom surface and x number of hollow microneedles with internaldiameter d protruding from bottom base surface wherein x is greater than3; ii) the hollow microneedles have sharp cutting edge at distal end andproximal end opening on top surface; b) i) second array comprises onebase plate with top and bottom surface and x number of solidmicroneedles with diameter 5 percent or more smaller than d andprotruding from bottom base surface wherein x is greater than 3; ii) thesolid microneedles have non-cutting surface at distal end; iv) thematrix arrangement of needles is identical to first array needles; c) i)the cartridge comprises a base plate with top and bottom surfaces with xnumber of holes in both surfaces and x is greater than 3; and thearrangement and internal diameter of holes is same as first array.

In an embodiment, a drug delivery microneedle array device comprises; 3or more hollow microneedles arranged in an array format attached to asolid base plate and each microneedle has an piercing element at thedistal end that is configured to pierce the human or animal skin tissueand proximal end is attached to a base plate wherein the proximal end ofhollow microneedle is exposed on the base plate; the hollow microneedlespace in atleast 3 microneedles is partially or completely occupied by asolid sustained drug delivery unibody microimplant composition whereinthe unibody composition is devoid of piercing element and the unibodymicroimplant composition can be pushed in the skin tissue by applicationof gas or liquid pressure or mechanical force.

In an embodiment, a drug delivery microneedle array device comprises twomicroneedle arrays wherein one array comprises an injectable unit thatcomprises x number of hollow microneedles attached to a base plate andarranged in an array format wherein x is greater than 3 and each hollowmicroneedle has a piercing element at the distal end that is configuredto pierce the human or animal skin tissue and proximal end is attachedto a base plate wherein the hollow cavity of microneedle is exposed onthe base plate; the hollow microneedle space is partially or completelyoccupied by a solid sustained drug delivery unibody microimplantcomposition; the other array has Y number of solid non-piercingmicroneedles attached to a base plate and arranged in the same arrayformat as hollow microneedle array and y is less than or equal to x; andthe solid microneedles can be inserted in hollow space of hollowmicroneedle array via exposed hollow cavities on the base plate and canpush the unibody microimplant composition out of the hollow cavity andin to skin tissue. In an embodiment, a sustained drug delivery unibodymicroimplant array composition in a live or bioprosthesis tissue caninclude: i) each microimplant has a non-piercing edge at proximal end;ii) the number of microimplants in the array is greater than or equal to3; ii) each microimplant is separated by a spacing of by more than 10microns; iii) each microimplant is implanted at a depth of 50 microns to5 mm in the tissue; iv) and the volume of each microimplant is less than0.05 ml. In an embodiment, a method for forming drug deliverymicroimplant array formed in a live or bioprosthesis tissue includes: i)forming 3 or more artificial cavities in the tissue in array format; ii)each artificial cavities is separated by a spacing of between 10 micronsto 5 mm; iii) partially or completely filling 3 or more cavities with apolymer solution having an effective amount of polymer dissolved in abiocompatible, water-soluble organic solvent; iv) precipitating thepolymer from the injected polymer solution in the cavities.

In an embodiment, a method for forming microimplant array in the live orbioprosthesis tissue comprises: a) forming 3 or more artificial cavitiesin the tissue in array format; b) filling the cavities partially orcompletely with an injectable fluid composition comprising a drugwherein: i) the number cavities formed is greater than or equal to 3;ii) each cavity has a volume of 1×10E-10 to 0.03 ml; iii) each cavity isseparated by a spacing of between 10 microns to 5 mm; v) each cavity isformed by displacing or destroying tissue or combination thereof.

In an embodiment, a microimplant array composition with live mammaliancells in a live or bioprosthesis tissue wherein: ii) the number ofmicroimplants implanted is greater than or equal to 3; ii) eachmicroimplant is separated by a spacing of between 10 microns to 5 mm;iii) each microimplant is implanted at a depth of 50 microns to 5 mm inthe tissue; iv) the volume of each microimplant is 1×10E-10 to 0.03 ml.

In an embodiment, a method for forming microimplant array with livemammalian cells in the live or bioprosthesis tissue comprises: a)forming array of 3 or more artificial cavities in the tissue; b) eachcavity has a volume of 1×10E-10 to 0.05 ml; iii) each cavity isseparated by greater than 10 microns; and iv) filling the cavities withan aqueous injectable composition comprising 1 to 10000000 live cellsper ml of injectable composition.

In an embodiment, a microimplant array composition comprises livemammalian cells formed in a live or bioprosthesis tissue wherein: i)each microimplant has 1 to 10000000 live cells; ii) the number ofmicroimplants in the array is greater than or equal to 3; ii) eachmicroimplant is separated by a by 10 microns; iii) each microimplant isimplanted at a depth of 50 microns to 5 mm in the tissue.

In an embodiment, a method for delivering an injectable composition at alocal site in a tissue comprises: providing an aqueous injectablecomposition having 1 to 1.0E 7 live mammalian cells per ml; andinjecting the injectable composition into the tissue at the rate of10-12000 injections per minute and/or at an amount of 1.0E-02 ml to1.0E-16 ml per injection

In an embodiment, a method of forming an implant in the tissue, themethod comprises: providing a polymer solution having an effectiveamount of polymer dissolved in a biocompatible, water-soluble organicsolvent and mixing the polymer solution with a biodegradable filler toform a suspension or emulsion; injecting the polymer solution in thetissue; dissipating the biocompatible, water-soluble organic solvent inthe tissue; and precipitating the polymer from the injected polymersolution so as to form the implant.

In an embodiment, a method of delivering a drug in a solid state formwithout forming a solution includes: a) providing 3 or more solidunibody microimplants comprising effective amount of drug encapsulatedor coated on a biodegradable unibody forming matrix; b) implanting 3 ormore implants simultaneously in a live tissue or bioprosthesis tissue toform a microimplant array c) each microimplant is separated by a spacingof between 10 microns to 5 mm; d) each microimplant is implanted at adepth of 50 microns to 5 mm in the tissue; f) the volume of eachmicroimplant is 1×10E-10 to 0.03 ml.

In an embodiment, a method of delivering a Botulinum toxin in a solidstate form without forming a solution comprises: a) providing 3 or moresolid unibody microimplants comprising effective amount of Botulinumtoxin encapsulated or coated on a biodegradable unibody forming matrixwherein each microimplant has at least 0.01 units of Botulinum toxin; b)implanting the drug in a live tissue or bioprosthesis tissue to form amicroimplant array; c) each microimplant is separated by a spacing ofbetween 10 microns to 5 mm; d) each microimplant is implanted at a depthof 50 microns to 5 mm in the tissue; iv) the volume of each microimplantis 1×10E-10 to 0.03 ml.

A method of delivering a vaccine in a solid state form without forming asolution, the method comprising: a) providing 3 or more solid unibodymicroimplants comprising effective amount of vaccine encapsulated orcoated on a biodegradable unibody forming matrix; b) implanting 3 ormore implants simultaneously in a live tissue or bioprosthesis tissue toform a microimplant array; c) each microimplant is separated by aspacing of between 10 microns to 5 mm; d) each microimplant is implantedat a depth of 50 microns to 5 mm in the tissue; iv) the volume of eachmicroimplant is 1×10E-10 to 0.05 ml.

In an embodiment, a method of delivering a bioactive compound comprisingiron in a solid state form without forming a solution includes: a)providing 3 or more solid unibody microimplants comprising effectiveamount of iron compound coated or encapsulated in a biodegradableunibody forming matrix; b) implanting 3 or more implants simultaneouslyin a live tissue or bioprosthesis tissue to form a microimplant array;c) each microimplant is separated by a spacing of between 10 microns to5 mm; d) each microimplant is implanted at a depth of 50 microns to 5 mmin the tissue; iv) the volume of each microimplant is 1×10E-10 to 0.05ml.

A method of treating nail infection, the method comprising: i) formingan array of 3 or more artificial cavities in the nail; ii) eachartificial cavities is separated by a spacing of between 10 microns to 5mm; iii) partially or completely filling 3 or more cavities with apolymer solution having an effective amount of polymer dissolved in abiocompatible, water-soluble organic solvent and an effective amountantifungal drug; iv) precipitating the polymer from the injected polymerin the cavities and entrapping the antifungal drug in the precipitatedpolymer.

c) the second array needles can be inserted in hollow cavity of firstarray via needle opening on top surface of first array via cartridgeholes after aligning center of needles of both the arrays and cartridgeholes.

In an embodiment, a process for sustained drug delivery in tissuethrough an array created in situ can include steps: creating anartificial porosity of predetermined geometric configuration anddimensions, filling the artificial porosity with fluid injectable drugdelivery compositions, releasing the drug in the surrounding tissue bydiffusion or biodegradation, wherein the artificial porosity comprisesof a plurality of cavities having predetermined volume, shape andsurface area and created in predetermined quantity and pattern with aspecific tissue area and depth.

A process can include the artificial porosity being created by meanssuch as displacing or destroying the tissue, including water jetdrilling based methods, ultrasonic energy based methods, particlebombardment based methods, laser based methods, oscillating needle,mechanical drilling and microneedle array based methods.

A process can include the array having a configuration of at least a 2×2matrix.

A composition can be provided for local sustained delivery of a drugpresent in the form of a micro array in a tissue, wherein suchcompositions comprise of drug encapsulated microimplants that are madeor implanted in situ and deliver the drug in a sustained manner.

A microimplant array can be created in an artificial cavity in a tissuecomprising a combination of a biodegradable polymer and a drug.

A microimplant array can be formed in a plurality of artificial cavitiesin a tissue comprising a combination of a hydrogel and live mammaliancells.

A device can be provided for creating a microimplant array for sustaineddrug delivery, wherein the device enables to form one, two, three, four,five, six or several microimplants in the tissue.

An “array-in-array” device can be provided for creating a microarray ofimplants for sustained drug delivery, wherein the device comprises of aplurality of first micro needle array and a plurality of second microneedle array, such that the plurality of first micro needle array isdisposed concentrically within the plurality of second micro needlearray.

A device can have the microneedle being hollow, solid or bioerodable.

A microneedle array patch can be provided for sustained drug deliverycomprising 4 or more hollow microneedles per centimeter square, whereinthe said microneedles cavities are loaded with microimplants forsustained drug delivery and each implant has a volume of 1×10E-10 to0.03 ml. A metal, plastic or ceramic hollow microneedle array patch canbe provided for sustained drug delivery or for mammalian cell deliverywith four or more hollow microneedles per cm² microneedles, wherein thesaid microneedles comprising a structure comprising a base at a proximalend and a vertex or tip with sharp edge that enables tissue insertion;and the hollow cavity of microneedle is partially or completely filledwith solid or hydrogel implant wherein the implant has a volume of1×10E-10 to 0.03 ml per needle.

A method of making a microimplant array for sustained drug delivery caninclude: providing a hollow microneedle patch with four or more hollowmicroneedles per cm² and cavity volume of 1×10E-10 to 0.03 ml perneedle; filling the hollow cavity with a biostable or biodegradablepolymer solution and drug; and removing the solvent from the solutionand precipitating the polymer and drug in the cavity; inserting thearray in the tissue and dispensing the implant from the needle cavity into the tissue.

A method of making an array of sustained drug delivery implants in thetissue can include: providing a hollow microneedle patch with four ormore hollow microneedles per cm² and cavity volume of 1×10E-10 to 0.03ml per needle; filling the hollow cavities of microneedles with abiostable or biodegradable polymer solid implant that can fit inside thecavity of the needle; insetting the needles of the array in the tissueand dispensing the implant from the cavity in to the tissue.

A method of forming an implant in the tissue can include: providing apolymer solution having an effective amount of polymer dissolved in abiocompatible, water-soluble organic solvent; injecting the polymersolution in the tissue using hollow microneedle array patch with four ormore hollow microneedles per cm²; dissipating the biocompatible,water-soluble organic solvent in the tissue; and precipitating thepolymer from the injected polymer solution so as to form the implant.

A method of making an array of implants with live mammalian cells in thetissue can include: providing a hollow microneedle patch with four ormore hollow microneedles per cm² and cavity volume of 1×10E-10 to 0.03ml per needle; filling the hollow cavities of microneedies with abiostable or biodegradable hydrogel implant comprising live mammaliancells that can fit inside the cavity of the needle; inserting theneedles of the array in the tissue and dispensing the implant from thecavity in to the tissue.

A microneedle array can include mammalian live cells and frozen matrixthat has a vertex or tip with sharp edge at distal end that enablestissue insertion; base at proximal end; wherein the frozen matrixcomprises cryopreservative.

An array in array device for sustained delivery of drugs or live cellsin tissue, the drug composition being in the form of a unibodymicroimplant, can include: a base array, the base array furthercomprising a base array plate, having a top surface and a bottomsurface, and a plurality of hollow microneedles provided in an arrayformat and protruding from the bottom surface of the base array plate,said hollow microneedles having a piercing element at the distal endthat is configured to pierce the skin tissue, a proximal end that isattached to the base array plate, and a hollow cavity capable ofcontaining a unibody implant, a plurality of guiding posts protrudingfrom the bottom surface of the base array plate; a plunger array, theplunger array further comprising a plunger array plate, having a topsurface and a bottom surface, and a plurality of solid microneedlesprovided in an array format on the bottom surface of the plunger arrayplate, said solid microneedles characterized by a non-piercing elementat the distal end, a proximal end that is attached to the plunger arrayplate, and capable of pushing the unibody implant contained in thehollow cavity of the hollow microneedles, a plurality of guiding holesprovided on the plunger array plate at predetermined locations; aplurality of spacer locks; wherein: the plunger array is capable ofcoaxial vertical movement within the base array, the plurality of solidmicroneedles of the plunger array is less than or equal to the pluralityof hollow microneedles of the base array, the base array and the plungerarray being vertically aligned above the tissue, and dimensionallycharacterized such that the plurality of solid microneedles of theplunger array is smoothly inserted in the plurality of the hollowmicroneedles of the base array.

A device for sustained delivery of drugs or live cells in tissue, thedrug composition being in the form of a unibody microimplant, caninclude: a plurality of hollow microneedles arranged in an array format,a base member, wherein each microneedle has an piercing element at adistal end that is configured to pierce the tissue and a proximal endthat is attached to the base member, wherein the hollow cavity ofmicroneedle is exposed on the base plate; the hollow microneedle spaceis partially or completely occupied by a solid sustained drug deliveryunibody microimplant composition wherein the unibody composition isdevoid of piercing element and the unibody microimplant composition canbe pushed in the skin tissue by application of gas or liquid pressure ormechanical force.

An array of unibody microimplants, disposed within a live orbioprosthesis tissue at a predetermined depth and in a predeterminedpattern, can include: said array comprises of a plurality of unibodyimplants characterized by non-piercing edges, the plurality of unibodymicroimplants is at least three, and has a separation distance between apair of unibody microimplants in the range of 10 microns to 5 mm, thepredetermined depth is in the range of 50 microns to 5 mm, the volume ofeach unibody microimplant is at most 0.05 ml, and the composition ofeach unibody microimplant is obtainable from a combination of abioactive agent, live cells, one or more biodegradable polymers, aporous coating material, a crosslinking agent, a hydrogel, an injectablefluid and a visualization agent.

An array of unibody microimplants, disposed within a live orbioprosthesis tissue at a predetermined depth and in a predeterminedpattern, can include: said array comprises of a plurality of unibodymicroimplants for sustained delivery of a bioactive agent in a solidstate form, and each unibody microimplant is characterized bynon-piercing edges, the plurality of unibody microimplants is at leastthree, and has a separation distance between each unibody microimplantsin the range of 10 microns to 5 mm, the predetermined depth is in therange of 50 microns to 5 mm, the volume of each unibody microimplant isat most 0.05 ml, and the composition of each unibody microimplant isobtainable from a combination of a protein based bioactive agent, abiodegradable polymer, a porous coating material, a crosslinking agent,a hydrogel, an injectable fluid, a visualization agent and an encodingagent.

A ready-to-inject microneedle array device can include a plurality ofhollow microneedles arranged in an array format, and preloaded with acorresponding plurality of unibody microimplants, wherein: each unibodymicroimplant is obtainable from a combination of a protein basedbioactive agent, a biodegradable polymer, a porous coating material, acrosslinking agent, a hydrogel, an injectable fluid, a visualizationagent and an encoding agent, the plurality of microneedles is at leastthree, the microneedles are inserted in into the tissue to a depth, andthe volume of each unibody microimplant is at most 0.05 ml.

A method for delivering a vaccine or a protein based drug in a solidstate form, in a live tissue, as an array of unibody microimplants caninclude: providing a drug composition in a biodegradable microneedlearray, creating an artificial porosity of predetermined geometricconfiguration and dimensions, filling the artificial porosity with aplurality of unibody microimplants disposed within the biodegradablemicronedle array, releasing the drug composition in the live tissue bydiffusion or biodegradation, wherein the artificial porosity comprisesof a plurality of cavities having predetermined volume, shape andsurface area and created in predetermined quantity and pattern with aspecific tissue area and depth.

A method for delivering live cells as an array of unibody microimplants,in a live tissue, can include steps: providing a dissolvable, hollowmicroneedle array, filling the cavities of hollow microneedle array withhydrogels comprising live cells, inserting the dissolvable hollowmicroneedle array containing live cells inside the tissue; pushinghydrogels comprising live cells out of the hollow microneedle array intothe tissue; removing the hollow microneedle array from the tissueleaving behind the hydrogel array comprising live cells inside thetissue.

A method for sustained drug delivery in tissue through an array createdin situ can include steps: creating an artificial porosity ofpredetermined geometric configuration and dimensions, filling theartificial porosity with fluid injectable drug delivery compositions,releasing the drug in the surrounding tissue by diffusion orbiodegradation, wherein the artificial porosity comprises of a pluralityof cavities having predetermined volume, shape and surface area andcreated in predetermined quantity and pattern with a specific tissuearea and depth.

A microneedle array patch for sustained drug delivery comprising threeor more hollow microneedles per centimeter square, wherein the saidmicroneedles cavities are loaded with microimplants for sustained drugdelivery and each implant has a volume of 1×10E-10 to 0.05 ml. A metal,plastic or ceramic hollow microneedle array patch for sustained drugdelivery or for mammalian cell delivery with four or more hollowmicroneedles per cm² microneedles, can include said microneedles havinga structure comprising a base at a proximal end and a vertex or tip withsharp edge that enables tissue insertion; and the hollow cavity ofmicroneedle is partially or completely filled with solid or hydrogelimplant wherein the implant has a volume of 1×10E-10 to 0.05 ml perneedle.

Materials and Methods

Microneedle array are purchased from AdminMed (Sunnyvale, Calif.),Micropoint Technologies Pte Ltd. Singapore or Amazon (UPC code601913872222). Tissues like bovine pericardium, porcine pericardium,porcine submucosa, porcine aortic root, porcine meniscus tissue, porcinecornea and bovine thoracic arterial tissue, bovine cornea, porcine bloodand porcine plasma are acquired or purchased from commercial sourcessuch as Animal Technologies, Tyler, Tex. or obtained from local abbotairor slaughter house. Submucosa tissue is obtained after cleaning andremoval of tunica mucosa, muscular tissue and serous layers from thefresh porcine, sheep or bovine small or large intestinal tissue.Polyethylene glycol can be purchased from various sources such as, byway of example, and not limitation, Nektar Therapeutics (formerlyShearwater Polymers), Dow Chemical's (Union Carbide), Fluka andPolysciences. Various protein crosslinkers especially diacid or polyacidn-hydroxysuccinimide esters or n-hydroxysulfosuccinimide esters may bepurchased form Sigma-Aldrich or Thermo Fisher Scientific (Pierce).Multifunctional hydroxyl and amine-terminated polyethylene glycol arepurchased from Nektar Therapeutics, Dow Chemicals, Huntsman Corporationand Texaco. Amine-terminated polyethylene glycols also can besynthesized using methods known in the prior art or may be purchasedfrom Aldrich (Jeffamine® ED-2003). Other specialized polyethylene glycolderivatives may also be purchased or custom synthesized from NektarTherapeutics, BOC Sciences, Shirley, N.Y., or Laysan Bio, Inc. AL.DL-lactide, glycolide, caprolactone and trimethylene carbonate can beobtained from commercial sources like Purac, DuPont, Polysciences,Aldrich, Fluka, Medisorb, Wako and Boehringer Ingelheim.N-hydroxysulfosuccinimide can be purchased from Pierce or Aldrich. Allother reagents, solvents are of reagent grade and can be purchased fromcommercial sources such as, by way of example, and not limitation,Polysciences, Fluka, ICN, Aldrich and Sigma. Most of thereagents/solvents are purified/dried using standard laboratoryprocedures such as, by way of example, and not limitation, described byPerrin et al. Small laboratory equipment and medical supplies can bepurchased from Fisher or Cole-Parmer. Cell culture experiments areperformed using a standard mammalian tissue culture laboratory ormicrobiology laboratory capable of handling and growing mammalian andhuman cell cultures.

General Analysis

Chemical analysis such as, by way of example, and not limitation,structural determination is done using nuclear magnetic resonance(proton and carbon-13) and infrared spectroscopy and mass spectrometry.High-pressure liquid chromatography or UV-visible spectrophotometry isused to determine drug elution profiles. Gel permeation chromatographyis used for molecular weight determination. Thermal characterizationsuch as, by way of example, and not limitation, melting point, shrinktemperature and glass transition temperature is done by differentialscanning calorimetric analysis. The aqueous solution properties such as,by way of example, and not limitation, self-assembly, micelle formation,and gel formation are determined by fluorescence spectroscopy,UV-visible spectroscopy and laser light scattering instruments. Drugrelease studies are conducted in PBS under sink conditions at 37 degreeC. and the drug elution is monitored by HPLC or UV-VISspectrophotometer.

General methods about biodegradable microparticles and microspheres canbe found in relevant prior art. Some examples of biodegradablemicroparticles and microspheres can also be found in previously filed USprovisional applications U.S. Provisional Patent Application No.62/378,662 filed on Aug. 23, 2016 and U.S. Provisional PatentApplication No. 62/363,839 filed on Jul. 19, 2016 and U.S. Pat. No.9,072,678 cited herein for reference only.

Biodegradation and Biocompatibility of Implants

In vitro degradation of the polymers is monitored gravimetrically at 37degree C., in aqueous buffered medium such as, by way of example, andnot limitation, phosphate buffered saline (pH 7.2).

In vivo biocompatibility and degradation life times are assessed aftersubcutaneous implantation of tissue samples. The implant is surgicallyimplanted in the animal body. The degradation of the implant over timeis monitored gravimetrically or by chemical analysis. Thebiocompatibility of the implant is assessed by standard histologicaltechniques.

Example 1

Preparation of In Situ Implant for Sustained Drug Delivery UsingBiodegradable Filler

Use of Filler in Injectable Polymer Solution Systems

Use of Magnesium Carbonate as an Exemplary Biodegradable InorganicFiller.

100.2 mg PDLG 5002 polymer is dissolved in 1.0 ml DMSO. 10 microlitersof methylene blue stock solution (10 mg methylene blue in 2 ml DMSO) isadded to the polymer solution as a colorant. Infusion solutions areprepared using the following method: 15.2 mg bupivacaine hydrochlorideand 0.50 ml polymer solution are mixed until drug is completelydissolved. 50 mg magnesium carbonate powder (fine powder sieved tocollect fraction below 300 microns in size) is added as a biocompatibleand biodegradable filler in the drug solution and the mixture isvigorously vortexed for 5 minutes. The magnesium carbonate suspension isinfused/tattooed using an oscillating needle in 1 cm square area ofsheep skin tissue. Excess solution from the tattooed surface is wipedoff. The light blue tattoo with magnesium particles is clearly seen theby unaided naked eye.

Similarly, a polymer solution without magnesium carbonate is preparedand infused/tattooed in the sheep skin tissue as above. The infusedtissue sample is cut and used in controlled release experiment.

The cut samples are placed in 3 mL of PBS (pH 7.4). The drug eluted PBSis collected at several time points. Fresh PBS is added to replace theeluted PBS solution. The collected PBS is analyzed using a UVspectrophotometer to determine the drug concentration. Cumulative totaldrug released from the tissue is plotted against elution time and drugelution profile is shown in FIG. 21B. The sample with magnesiumcarbonate showed much longer sustained release as compared to sampleswithout magnesium carbonate.

Example 2-A

Preparation of in situ implant using biodegradable polymeric particulatefiller.

Use of polymeric particulate filler in a biodegradable polymer solutionin water miscible solvent.

Use of polyglycolic acid (PGA) microparticles as an exemplarybiodegradable polymeric filler in a biodegradable polymer solution inwater miscible solvent.

200.1 mg PDLG 5002, 2.0 ml polyethylene glycol dimethyl ether and 20microliter methylene blue stock solution (as a colorant) are mixed untilpolymer is completely soluble in the solvent. 15.1 mg bupivacaine baseand 0.5 ml polymer solution as above is mixed until complete solution.The solution is sterile filtered and is then mixed with 20 mg sterilePGA microcylinders (150 microns in diameter and 200 microns length),prepared by cutting the fibers/filaments/threads. The PGA particles areinsoluble in the polymer solution and form suspension upon vigorousmixing. 0.3 ml of the suspension is injected into chicken leg muscleusing standard 3 ml syringe and 16 gauge needle. The polymerprecipitates in the injected area leaving behind precipitated polymerentrapping drug and PGA microparticles.

In a similar experiment, microcylinders made using commercial coloredcatgut suture thread (Ethicon, chromic gut, colored, suture USP size6-0) and length 100 microns are used in place of PGA particles. Thecolor of particles may be used as a colorant in place of methylene blue.The rifampin coated microspheres incorporated in thermosensitive gel inExample 10H can be considered as biodegradable filler incorporated inthermosensitive gel. Similarly, inorganic filler such as magnesiumcarbonate may be added as a filler in thermosensitive gel precursorsolution prior to injection and gelling.

In another illustrative example, inorganic biocompatible, biodegradablefillers such as magnesium carbonate, calcium sulfate, and the like maybe mixed with crosslinkable polymer solution such as PEG35K-lactate-acrylate macromonomer solution (Example 10D) or PEG10KARMglutarate NHS ester and trilysine (Example 10E) are polymerized andcrosslinked with light to entrap the filler in the gel. The entrappedfiller can be used to alter the local chemical/physical environment ofthe crosslinked gel (pH, osmolality, surface area and the like) whichmay help to tune the sustained drug release of drugs (FIG. 19A).

Example 2-B

Use of polymeric particulate filler in a biodegradable polymer solutionin water miscible solvent.

Use of polyvinyl alcohol or protein (collagen or albumin) microparticls.

50.4 mg PDLG 5002, 0.5 ml polyethylene glycol dimethyl ether (PEGDME) asa polymer solvent 5.0 microliter methylene blue solution as a colorant(10 mg methylene blue dissolved in 2.0 ml DMSO) are mixed until polymeris completely dissolved. 7.5 mg Bupivacaine base (approximately 30percent of polymer weight) and 0.25 mg polyvinyl alcohol (PVA) powder asa biodegradable filler (sieved, PVA particle size less than 300 microns)are added to 0.25 ml polymer solution as above. The filler weight isapproximately same as PDLG polymer weight (1:1). The PVA powder isinsoluble in polymer solution but the drug Bupivacaine base is soluble.The suspension is infused in the bovine pericardium tissue in 1 squarecentimeter area using oscillating needle to form a drug deliveryarray/implant. The infused polymer precipitates in the hydrated tissueafter dispersion of PEGDME solvent in the tissue, entrapping drug inPDLG polymer. PVA filler is also entrapped. The PVA filler is believedto provide large surface area for polymer precipitation. The controlsolution (without Bupivacaine base) with PDLG and PVA in DMSO as aboveis also infused in bovine pericardium tissue. The release of Bupivacainebase is monitored from control and treated tissue. In this illustrativeexample PVA acts as a biodegradable insoluble filler.

In another modification of above embodiment, PVA filler is replaced withcrosslinked gelatin microspheres (size less than 300 microns).Crosslinking prevents solubility in common water miscible organicsolvents used for biodegradable polymer solution preparation. In anothermodification of above embodiment, PVA filler is replaced withcrosslinked PEG based biodegradable microspheres (size less than 300microns) made by polymerization of PEG-hydroxyacid based macromonomerssimilar to described in Example 10D. Crosslinking of PEG based hydrogelparticles prevents their solubility in common water miscible organicsolvents used for biodegradable polymer solution preparation. Thebiodegradable bonds such as ester bonds present in the crosslinkedparticles enables biodegradation.

In another modification of above embodiment, PVA filler is replaced withcrosslinked PEG based biodegradable microspheres (size less than 300microns) made by condensation polymerization of PEG based precursorswith nucleophilic and electrophilic groups, similar to described inExample 10E.

Example 3

Preparation of Colored and Drug Encapsulated Microspheres

Preparation of synthetic biodegradable polymer encapsulated microspherescomprising rifampin.

0.5 g Poly(lactide-co-glycolide) copolymer (PLGA)(PURAC Biochem,Netherlands, PDLG 5002 polymer) is dissolved in 4.5 ml ethyl acetate tomake approximately 10 percent solution. In a 15 ml glass vial, 10 mg ofRifampin is added to 1 ml PLGA ethyl acetate solution and vortexed untilcomplete dissolution of the drug. In a 50 ml beaker, 5 ml PVA solution(1 percent in distilled water, Sigma Aldrich, catalog no P8136,30000-70000 g/mol, 87-90 percent hydrolyzed) and magnetic stir bar areadded. The solution is stirred and while stirring, drug solution isadded dropwise using disposable plastic dropper. After completeaddition, the solution is transferred to 50 ml PP centrifuge tube andvortexed for 2 minutes. The drug suspension is then added to 40 ml PVAsolution stirred vigorously using magnetic stirrer in a 250 ml beaker.The stirring is continued overnight to remove ethyl acetate. Next day,the red drug suspension is transferred to 50 ml polypropylene centrifugetube (approximately 35 ml suspension). The suspension is centrifuged at2500 rpm for 10 minutes. The supernatant is removed and the rifampinpellet is resuspended in 35 ml distilled water and vortexed vigorouslyfor 2 minutes. The suspension is observed under microscope and the redcolored microspheres are clearly observed floating in the suspension.The process is repeated one more time and supernatant is removed almostcompletely. The microspheres are lyophilized and recovered as redcolored powder.

In another embodiment, 1 g PDLG 5002 polymer and 0.2 g rifampin isdissolved in 100 ml dichloromethane and the mixture is spray dried usinga standard lab based spray drier. The rifampin encapsulated microspheresare collected, characterized and stored at −80 degree C. until use.

Example 4

Preparation of Hydrogel Based Biodegradable Polymers

Protein Based Colored Biodegradable Microspheres.

0.2 g gelatin is dissolved in 1.8 ml 0.1 M MES buffer, PH 5.5. To thissolution, 100 mg eosin Y, 0.3 g n-hydroxysuccinimide and 0.3 g EDC areadded. After complete dissolution, the mixture is added to 100 mlmineral oil and stirred vigorously. The crosslinking reaction andstirring is continued for 12 hours. The gelatin microspheres areseparated by filtration and washed with hexane to remove traces ofmineral oil. The eosin stains as well as forms covalent links to thegelatin. Using a similar procedure, fluorescein (free acid form) can bechemically bound to the crosslinked albumin to make it fluorescent.

Example 5

Preparation of Hydrogel Based Biodegradable Microspheres with Drugs

1 g bovine albumin is dissolved in 3 ml PBS. To this solution 100 mg ofchlorhexidine gluconate is added. The solution/suspension is stirred andtransferred to 10 ml syringe with 22 gauge needle. The albumin solutionis dispensed from the syringe or sprayed from a sprayer into 1000 mlliquid nitrogen. The frozen droplets are collected. Liquid nitrogen isevaporated. The frozen microspheres are exposed to 0.25 percentglutaraldehyde solution at zero degree C. for 30 minutes in PBS pH 7.2to crosslink albumin. The crosslinked microspheres are washed with PBS 3times and then lyophilized. The crosslinked hydrogel microspheres may bevacuum dried at room temperature to dehydrate them. The dehydratedmicrospheres can have smaller size or more relative to its hydrated sizeand can regain the original size by abortion of water.

In a modification of above procedure, the albumin is crosslinked with 20mg/ml in PBS pH 7.2 disulfosuccinimidyl suberate for 12 h.

In a similar experiment, chlorhexidine is replaced by 100 mg rifampin tomake rifampin loaded colored microspheres.

Example 6

Preparation of porosity in the tissue or model tissue like materialssuch as gelatin gel.

Preparation of porosity using metal microneedle array.

Porosity creation in sheep dermal skin.

Example 6A

Artificial Porosity Created Using a Metal or Plastic Solid MicroneedleArray

A sheep skin is freshly procured from the local abattoir. One side isshaved to remove all the hairs and other side is (dermis layer side) isused for making artificial porosity. AdminStamp 600 Microneedle ArrayDevice is used to create micropores in the skin tissue. This device has187 five hundred microns tall stainless steel microneedles on 1 squarecentimeter (circular shape). Briefly, the needles are applied (needleare positioned perpendicular to the facing skin surface) on the dermisside of the skin tissue. The stamp is rotated 180 degrees three times tomake a circular hole in the skin tissue. The same area is treated threetimes. This stamped area in the skin created 500 microns tall 187cylindrical holes in the tissue. These artificial cavities are usedfurther to infuse injectable compositions.

In another experiment, a gelatin gel is cast in a plastic petri dish.Briefly, 10 g food grade gelatin (Knox Original Unflavored Gelatin) isdissolved in 90 g distilled water and the solution is heated to 70degree until complete solution. The solution is poured in the petri dishand the dish is cooled in refrigerator. Upon cooling, the gelatin isconverted into soft transparent gel and is used as a model flesh/tissuematerial to optimize porosity creation experiments. Due to itstransparency, it may be desired in some cases to optimize porositymethod development and injectable formulation development. AdminStamp600 Microneedle Array Device is used to create 500 microns tall 187cylindrical holes.

In another similar experiment, AdminStamp 777 Microneedle Array Deviceis used. This device has 121 seven hundred micron tall microneedles onone square centimeter circular microneedle array. In another similarexperiment, eighty-five 800 micron tall microneedles arranged in onesquare centimeter circular microneedle array is used. Yet in anotherexperiment forty-three 1100 microns tall microneedles arranged in onesquare centimeter circular microneedle array are used. Yet in anotherexperiment thirty-one 1400 microns all microneedles arranged in onesquare centimeter circular microneedle array are used. This exampleshows different types of cavities with various cavity densities anddifferent heights can be created by using commercially available metalmicroneedle arrays.

Example 6B

Artificial Porosity Created Using a Hollow Stainless Steel MicroneedleArray

A 3 by 3 stainless steel hollow microneedle hub is obtained fromMicropoint Technologies Pte Ltd. (Singapore) (referred as 33 MP). Thehub has a square base pyramidal shaped needle with height of 1000microns, rectangular base 300×300 microns, internal diameter 150microns, needle pitch 1000 microns and needle's center-to-center spacingis 0.63 mm. The hub has a stainless steel female Luer lock connectorwhich can be connected to a syringe with male Luer Lock connector. Allthe needles in the device can inject liquid when injected via Luer Lockand syringe. The empty hub is connected to a syringe which is filledwith a saline. The hub is inserted in the sheep skin tissue to createarray of 3 by 3 holes in the tissue. Total 9 holes created in oneinsertion. The same hub is used to make two sets of 3 by 3 holes on thesame tissue. Total 27 holes are created. The holes had same dimensionsas external shape of the needles on the hub. To visualize the holes,gelatin gels are used instead of skin tissue to make the cavities. Thesize on the holes in gelatin is measured using the optical travellingmicroscope. In a similar experiment, a hub with 10 by 10 array with 700micron needle height is used for injection.

Example 6C

Artificial Porosity Created Using Dissolvable Array

Casting of Dissolvable Array Using Silicone Rubber Mold.

Silicone base MPatch™ Microneedle templates are procured from MicropointTechnologies Pte Ltd. (Singapore). The mold has followingcharacteristics: 20 mm dia and 4 mm height. 10 by 10 microneedle arrayholes, 700 microns cavity height (square pyramid shaped cavities) with200 by 200 microns base, 500 microns pitch and distance between eachneedle is 500 microns (center to center). The mold is washed with mildsoap and dried under nitrogen. 0.5 g low viscosity carboxymethylcellulose sodium salt (CMC, Sigma Catalog number C5678) and 1 mg ofsodium fluorscein as a colorant/or florescent agent are dissolved in 1.5ml distilled water. The polymer is added in several small quantity stepsto dissolve it completely (24 to 48 hours for dissolution). The solutionis centrifuged to 2000 g for 40 minutes to remove entrapped air underclosed and humidified conditions to prevent moisture loss. The air freesolution is transferred on silicone mold surface. The mold and solutionis spun for 4700 rpm to drive the solution in the cavities and fill themcompletely. The solution is dried while rotating for 8 h or air dried atroom temperature (30%-45% RH) for 4-48 hours. After drying, an adhesivetape is applied on the mold (base of the needles) and the array isremoved gently from mold. The array is then inserted in the sheep skintissue or gelatin gel as mentioned previously. The polymer in the arraydissolves in the tissue fluid which can be aspirated or suctioned. Thecavities created can be used for filling with various injectablematerials as described previously. In this example, it is believed thatthe needle displaces the tissue to create a space for itself.

In another modification of this embodiment, CMC solution is replacedwith polyvinylpyrrolidone (molecular weight 55000) solution (25percent). In another modification maltose is used instead of CMC. Theadvantage with sugar is that it is dissipated by the tissue and noremoval of polymer may be needed prior to cavity filling.

In another modification of this embodiment, 20 mM PBS (Ph 7.4) solutioncontaining 10 percent dimethyl sulfoxide as cryopreservative is degassedby freezing and thawing under vacuum is used to fill the mold cavitywith little excess to form a 3 mm thick layer on top of mold. The waterand mold is centrifuged and while being centrifuged, the mold and wateris cooled below −80 degree to form frozen solid. The mold is separatedfrom the needles (in cold chamber below −10 degree C.) to preventmelting of frozen microarray needles. The top 3 mm layer forms a blockof ice on which ice needles are attached. The array in frozen state isapplied on top of porcine dermal tissue held at 37 degree C. to mimicbody temperature. The frozen block helps to apply pressure and push theneedle in the skin. The frozen needles penetrate in the tissue and meltinside the tissue first due to higher temperature of the tissue and formcavities inside the tissue. The frozen block holding the needle does notmelt during the experimental time frame due to larger volume/mass andnot in direct contact with the tissue. After melting the needles, thefrozen block is removed. The melted composition or PBS is dispersed inthe tissue. The cavities formed can be used to fill injectablecompositions. Other aqueous solutions such as distilled water,osmotically balanced solutions such as 0.9 percent sodium chloridesolution in water, HEPES buffer, triethanol amine buffer solutions pH7.4, solutions containing amino acids, tissue culture medium and thelike may be used in frozen form or state to penetrate the tissue andform cavities. The use of cryopreservative like DMSO is preferred whenworking with mammalian cells. Alternatively, methods described by B.Bediz et al., “Dissolvable Microneedle Arrays for Intradermal Deliveryof Biologics: Fabrication and Application” Pharm Res., volume 31(1),page 117-135, cited herein for reference only, may also be used.

Hyaluronic based dissolvable microarray can also be purchased fromMicropoint Technologies Pte Ltd. (Singapore). MPatch™ Mini is hyaluronicacid based microneedle patch along with its proprietary applicator canalso be used in making micro-cavities in the skin tissue. Uponapplication on skin tissue, the patch usually dissolves in 1-10 minutesdepending on the location producing micropores in the skin tissue.

Example 6D

Artificial Porosity Created Using Oscillating Needle.

An oscillating coring needle (500 micron internal cutting diameter,stainless steel) is attached to a tattoo machine or permanent makeuptattoo machine. The needle is oscillated at 10-12000 times per minute,typically at 600-1000 times per minutes with penetration distance keptat 500 microns. The machine is used on a porcine dermal tissue or bovinepericardium to create pores. Briefly the tattoo machine is set to goonly 500 micron dip in the tissue. The skin tissue is applied with alubricant such as vitamin E and the needle is slowly moved on skintissue. As needles goes in and out of skin tissue, it creates 500microns diameter size and 500 dip holes in the tissue. The needle ismoved on the tissue surface so that each insertion point is different onthe tissue surface and the movement is followed predetermined matrixpattern such as 10 by 10 matrix.

Example 6E-1

Artificial Porosity Created Using Mechanical Drill

In this method, a micro drill bit is used to create 2 by 2 arraycavities using a drilling method. Hard tissue such as bone, skull ornail may be drilled to create artificial porosity of desired size andshape. A 1/64 size micro drill bit is attached to a standard drillingmachine. A portion of cut human nail is used as model substrate tocreate holes. 4 holes (about 400 microns diameter) and 200 microns deepare drilled into nail. In another example, a fresh cow femur bone isisolated from local slaughter house. A 1/32 inch size micro drill bitand drill machine is used to drill 3 by 3 array (9 holes separated by 2mm) holes on bone surface. The hole diameter is about 800 microns anddepth is 200 microns.

Example 6E-2

Artificial Porosity Created by Syringe Needle

In another embodiment instead of drill, a fine syringe needle is usedmanually to create a cavity array in the tissue. Approximately 15 mm by15 mm dry pericardium tissue is used to make 4 by 4 array of cavities. A24 gauge needle is used to core approximately 1 mm dip cavity by hand inthe tissue. Total 4 cavities are made, 2 mm apart from each other, alongthe length of the tissue to make one row of cavities. Total four rowsare made, 2 mm apart each to make a 4 by 4 cavity array where eachcavity is separated by 2 mm. FIG. 8D shows image of a bovine pericardialtissue with cavities in 4 by 4 array format. Manual method for cavitycreation can be useful but may not preferred where large number ofcavities are needled. Variables like the size of cavity, number ofcavities made, distance between each cavity, needle size used, depth ofcavity can be varied to obtain a suitable array structure. In anotherillustrative embodiment, instead of cavities, holes are created in thetissue in 4 by 4 matrix pattern. In this case tissue thickness (around 1mm) is the height of cavity and diameter is same as external diameter of24 gauge needle (approximately 565 microns).

Alternatively, a coring needle may be manually used to core holes. Amodified breast biopsy or coring needle and instrument is used to create2 by 2 array holes (4 holes, 500 microns diameter and 1000 micron dip)on 1 cm square rectangular area.

Example 6F

Artificial Porosity Created Using Laser Irradiation.

A bovine pericardium tissue or porcine dermal tissue is used to createlaser irradiated porosity. Harmony Elite Laser machine from AlmaLasers™, Buffalo Grove Ill., US is used in creating porosity. Themachine parameters are set using manufacturer recommendations andliterature references (E. H. Tudor et al., Table 1) for similar type ofmachine. Briefly following experimental parameters are used to createporosity (array of cavities) in the pericardium or dermal tissue.Wavelength 2940, spot size 225 microns, density 5%, power levels1.15-2.22 W, pulse durations 50-225 microseconds, pulse repetition rates100-500 Hz, and 2, 20, or 50 stacked pulses. These conditions resultingin pulse energies of 2.3-12.8 mJ/microbeam and total energy levels of4.6-640 mJ/microchannel. Variables like total power, spot size andshape, pulse repeating rate, number of stacks and the like are used tocreate array of 5 by 5 array cavities in one square centimeter separatedby 1 mm.

Example 7 Example 7A

Infusion of Injectable Compositions in the Artificial Porosity

Direct Infusion in the Artificial Cavities.

In this method, the injectable composition such as PLGA drugmicrospheres suspended in PBS buffer or PLGA solution in n-methylpyrrolidone (10 percent weight by weight, and 10 percent (relative toPLGA plus solvent weight) rifampin as drug and/or visualization agent,is applied or exposed on the newly created porosity in the tissue. Thecomposition is inserted in the porous space via capillary force action,gravity if incubated for sufficient amount of time (1 to 60 minutesincubation). The insertion can be further assisted by applying externalenergy/force or pressure. For example, a jet of nitrogen gas or carbondioxide gas via a 14 gauge syringe needle may be used to force the fluidcomposition in the cavity. A fiber or rod or array needles may be usedto sweep the injectable composition or mechanically “stuff” thecomposition in the cavities. Care is taken to ensure that polymer is notprematurely precipitated on the skin surface if used as a polymersolution. Upon insertion in the cavity, the composition can undergofurther transformation (either physical or chemical) which enables toentrap the drug and release it slowly over a period of time. In anothermodification, the PLGA solution as above is added in the cavity viasyringe and needle. The 30-34 gauge syringe needle is used to infusesolution in each cavity individually and excess solution is wiped offfrom the surface.

The solution may also be sprayed/atomized and the droplets may becollected in the cavities.

Example 7B

Creation of Artificial Porosity and Infusion of Injectable Compositionsin the Artificial Porosity.

Application of microneedle array via injectable composition layer onskin/tissue surface to create porosity and inject composition in theporosity at the same time.

In this method, injectable fluid composition such as PLGA solution withrifampin in NMP or rifampin microsphere suspension in PBS or in glycerolis applied on the tissue or skin to form a layer of injectablecomposition. 1 g of PLGA (50:50 lactide:glycolide, molecular weight15000 g/mole) polymer and 100 mg of moxifloxacin base and 0.1 mg ofmethylene blue as a colorant is dissolved in 9 g of methyl pyrrolidone(NMP). The solution is sterile filtered using 0.2 micron PTFE syringefilter. This solution is applied on the porcine or skin dermal tissue toform a liquid layer. The thickness of layer is may be greater than onemicron thick, typically 10 microns to 3000 microns thick. The porosityis creation is done through this layer. For example, a microneedle arrayAdminStamp 600 as an example or 3 by 3 stainless steel microneedle hubfrom Micropoint Technologies (33 MP) is applied on the skin through theinjectable composition layer. The needles of the array contact thesolution layer first and then skin tissue. As needles pierce the tissueand make the cavity, the injectable composition is carried along withthe needles and is deposited inside the newly created cavities. Theneedles are retrieved from the tissue leaving behind the injectablecomposition in the cavities. The process may be repeated multiple timesto fill the cavities completely. If the injectable compositions havedrug encapsulated microparticles, they are carried inside the cavity forlocal and sustained drug delivery. If the polymer solution is used as aninjectable composition, then the solvent from the composition in isdispersed in the tissue and polymer is deposited in the tissue. If thecomposition contains precursors of crosslinkable composition, then theprecursor composition undergoes in situ polymerization and/orcrosslinking inside the cavity and form a crosslinked material/hydrogelinside the cavity.

Example 7C

Creation of Artificial Porosity and Infusion of Injectable Compositionsin the Artificial Porosity at the Same Time.

Use of hollow needles array to create and fill porosity.

1 g of PLGA (50:50 lactide:glycolide, molecular weight 15000 g/mole)polymer and 100 mg of moxifloxacin base and 0.1 mg of methylene blue asa colorant is dissolved in 9 g of methyl pyrrolidone (NMP) or dimethylsulfoxide (DMSO). The solution is sterile filtered using 0.2 micron PTFEsyringe filter. The sterile solution is loaded into sterile syringe. Thesyringe is attached to sterile 3 by 3 stainless steel hollow microneedlehub from Micropoint Technologies (33 MP). The solution is infused in thesheep skin tissue and/or on gelatin gel. After inserting the needlearray in the tissue, the syringe solution is pushed inside the tissue.The array is pulled away from the tissue and the solution is pushed inthe cavities created by the array as it is pulled away. The excesssolution is wiped from the skin surface. The array is used a total of 3times at nearby tissue creating 27 cavities filled with the PLGAsolution. The blue solution in the cavity is clearly seen to the nakedeye. The polymer is precipitated in the cavity after NMP is dispersed inthe tissue. The precipitated polymer traps moxifloxacin in the polymer.The treated tissue is cut off and is used to monitor the release ofmoxifloxacin in 3 ml PBS at 37 degree over several hours. The PBS isexchanged at every time point of drug release study.

Another illustrative embodiment teaches that artificial space (cavity)is first created inside the tissue which is isolated by the needle wall.The space created by the array needle is then filled with injectablecomposition. When the 3 by 3 stainless steel microneedle hub is insertedin the tissue, it displaces the tissue and creates the space in thetissue. This space is isolated in the tissue from the needle wall. Theartificially created space in the tissue which is isolated by hollowneedle wall is then filled with injectable composition such as PLGAsolution with moxifloxacin. Upon removal of the needle, the polymerstays in the tissue cavity forming an array of implant (27microimplants) in situ. Other injectable compositions such as precursorsof crosslinkable compositions, drug encapsulated microparticles, neatliquid based compositions, low melting compositions may also be used inplace of polymer solution as described here.

Example 7D

Creation of Artificial Porosity and Infusion of Injectable Compositionsin the Artificial Porosity. Use of Dissolvable Microarray.

In this embodiment, a dissolvable microneedle array such as hyaluronicor sugar based microneedle array is used for creating and fillingcavity. 1 g of PLGA (50:50 lactide:glycolide, molecular weight 15000g/mole) polymer and 100 mg of moxifloxacin base and 0.1 mg of methyleneblue as a colorant is dissolved in 9 g of polyethylene glycol dimethylether, molecular weight 550 or ethyl acetate). The solution is sterilefiltered using 0.2 micron PTFE syringe filter. This solution is appliedon porcine dermal sheep tissue to create a liquid layer of 10 to 100microns thick. The dissolvable microneedle array is applied through thislayer. (caution, the solvent used for polymer solution must be anon-solvent for the microneedle array material. For example,carboxymethyl cellulose or polyvinyl pyrrolidinone based materials arenot soluble in ethyl acetate or polyethylene glycol dimethyl ether,molecular weight 550). The available list of polymer solvent can befound in Polymer Handbook. The solvent and needle material are removedby the tissue water by dissolution or evaporation process or combinationthereof. The polymer precipitates in the artificial cavities created bythe needle.

In another embodiment, 1 g of PLGA (50:50 lactide:glycolide, molecularweight 15000 g/mole, ester endcapped) polymer is dissolved in 9 g ethylacetate. The needles of the carboxymethyl cellulose microarray are thendip coated with the PLGA solution and the solvent is evaporated. ThePLGA solutions forms a coating on the array needle. The coated array isinserted in the tissue where water dissolvable portion of the array isdissolved upon insertion leaving behind the PLGA coated film and thespace created by the dissolved array. If needed PBS solution or otherbiocompatible aqueous buffer solution is used to dissolve away theneedle. The space created by microarray dissolution remains isolated inthe tissue due to PLGA film. This space is then filled with theinjectable compositions such as fibrin glue or liquid carriers likevitamin E acetate or sucrose acetate isobutyrate, thermosensitive gellike Pluronic gel and the like described in this invention.

Example 8

Compositions for Treating Iron Deficiency.

Infusion of iron based compositions in the tissue.

Venofer® or its generic version (iron(III)-hydroxide sucrose complex) isa 20 mg iron/ml solution is procured from local pharmacy. 0.2 ml of thissolution (0.2 mg) is tattooed into 2 square centimeter of sheep skintissue. Please refer to U.S. Pat. No. 9,345,777 for additional detailson the use of tattooing process to deliver drugs into tissue. Thetreated tissue showed red color after infusion into the skin tissue.

In another method, soluble ferric pyrophosphate is obtained fromSigma-Aldrich Inc. (St. Louis, Mo.). The powder is ground and sieved toobtained less than 300 micron particles. 150 mg of sieved powder issuspended in 1 ml glycerol or polyethylene glycol 400 as biocompatiblewater soluble carrier. The 0.2 ml suspension is tattooed into 2 squarecentimeter area of the tissue (15 mg per square centimeter).

In another variation of this process, the sieved particles (150 mg) aresuspended in one ml of PLGA (50:50 lactide:glycolide, molecular weight15000 g/mole, ester endcapped) solution in n-methyl pyrrolidone (NMP).Methylene blue (small amount) is used as a colorant. 0.2 ml of thesuspension is tattooed in sheep dermal tissue and blue color of thetattooed portion is clearly visible. The excess solution duringtattooing process is wiped off from the tissue. The PLGA undergoesprecipitation in the tissue after dissipation NMP in the tissue. Acontrol sample without ferric pyrophosphate is prepared and tattooed asdescribed above. The entrapped iron is released from the precipitatedpolymer in the skin tissue. The release of iron from the treated tissueis monitored at 37 degree C. for 5 days. The iron is analyzed using aspectrophotometric method wherein a collected sample is mixed with 1.6ml 10% hydroxylamine hydrochloride solution, 0.4 ml 0.2 M sodium acetatesolution and 2.0 ml 0.25% phenanthroline solution and diluted up to 10ml using distilled water. Absorbance is recorded at 515 nm. Thesustained release of iron from treated and control sample is provided inFIG. 20.

Silicone rubber based MPatch™ Microneedle templates are procured fromMicropoint Technologies Pte Ltd. (Singapore); 10 by 10 microneedle arraycavities with 700 microns needle height cavities with 200 microns by 200microns base and 500 microns pitch. The mold is washed with mild soapand dried under nitrogen and sterilized using 70 percent isopropanol. 15mg ferric pyrophosphate+0.5 ml 1% sodium hyaluronate are mixed, sterilefiltered and added to the mold cavities. The water is removed by airdrying and the microneedle array containing hyaluronic acid and ferricpyrophosphate is removed using a polyester tape at the base. FIG. 9B1shows the dissolvable array containing hyaluronic acid and ferricpyrophosphate (902). FIG. 9B2 shows the microscopic image of one of theneedles of the arrays showing sharp edges and needle point (903). Thearray is pressed against the skin tissue and the needle dissolved in thetissue releasing ferric pyrophosphate in the tissue.

In another variation, cylindrical microimplants comprising ferricpyrophosphate are created first and then implanted using hollowmicroneedle array or “array in array” device described in thisinvention. Briefly a finely sieved powder (size less than 300 microns)of ferric pyrophosphate and dextran are mixed and compacted to producemicrocylinders of various size suitable for inserting in base arraycavity of AIA device described in this invention. The dextran acts as abinder and helps to form a unibody microimplant structure uponcompaction under pressure which enables to inject the ferricpyrophosphate as a unibody implant. The dextran dissolves away in thebody leaving behind the ferric pyrophosphate for therapeutic effect.

Example 9

Compositions for Treating Nail Infection

Human nail fragments are procured from human volunteers. A 1/64 inchmicro drill bit is used to drill 3 cavities along the width of the nail.The nail cavities had depth around 20-100 microns depending on the nailthickness. Care is taken not to drill hole all the way through the nail.100 mg Terbinafine hydrochloride, and 0.9 g, PLGA (50:50,dl-lactide:glycolide) are dissolved in 10 ml ethyl acetate ortetrahydrofuran or acetone. The polymer solution is applied on thecavities on the nail. All cavities are filled completely by the PLGAsolution. The nail is allowed to dry in air overnight. The release ofdrug from the nail is monitored in vitro in 3 ml PBS (pH 3.0) for 7days. In a similar experiment, the cavities are created by the laserdrilling process as described before. The cavities created by laserdrilling are coated/filled with PLGA solution with antifungal drug asdescribed before.

In another embodiment, a 3 by 3 array hollow needle array (33 MP) isused to create cavity and fill the cavities at the same time. The arrayhas 1000 micron height. The array is applied with 700 micron PTFE spaceron top of the microneedles to limit the penetration of array to 300microns only. The PLGA solution with Terbinafine hydrochloride solutionis sterile filtered and injected via array to make cavities and fill thecavities at the same time. Excess of solution is wiped off from thesurface. The treated area is exposed to PBS solution for 10 minutes toaccelerate the precipitation process in the cavity. The deposited drugin cavities release the drug in a sustained manner. This array creates300 micron dip cavities in the nail. In another embodiment, part ofhuman nail is cut. 4 cavities in 2 by 2 array format are created using a24 gauge needle (average cavity diameter around 600-700 microns). A PLGAbased polymer solution with D and C violet as colorant and terbinafinehydrochloride as antifungal drug is then used to fill in the cavity.FIG. 16A shows a photographic image of part of human nail withartificially created cavities FIG. 16B shows FIG. 16A cavities filledwith PLGA based biodegradable composition with D and C violet ascolorant. FIG. 16C shows in vitro terbinafine hydrochloride (anantifungal drug suitable for treatment of fungal nail infection) releaseprofile from PLGA based experimental composition.

Example 10

Delivery of Injectable Synthetic Biodegradable Polymer Solution inTissue.

Example 10A

Formation of In Situ Biodegradable Microimplants in the Live Tissue orBioprosthesis Tissue.

Creating a porosity first and then filling the pores with injectablecomposition (polymer solution in the second step.

Part 1: Preparation of Sterile Injectable Synthetic BiodegradablePolymer Solution with Drugs Suitable for Injection in the Live Tissue.Use of Water Miscible Organic Solvent

In a 50 ml glass beaker, 20 ml dimethyl sulfoxide (DMSO), 1.8 g Poly(PLGA, polylactide-co-glycolide) (dl-lactide:glycolide (50:50),molecular weight 13000 to 20000 g/mole.) and 200 mg (approx. 10 percentloading relative to weight of the polymer plus drug) moxifloxacin baseand 1 mg of methylene blue as a colorant are mixed until homogeneoussolution. The solution in the beaker is sterile filtered (filter hasPTFE membrane and polypropylene housing which is not affected by theDMSO solvent). The sterile filtered solution is used an injectablesolution to fill artificial cavities.

Part 2: In Situ Delivery of the Polymer Drug Solution Using Solid MetalMicroarray

About 2 cm by 2 cm portion rat back skin is shaved to remove hairs.Iodine solution is applied to sterilize the area. Sterile filteredvitamin E acetate oil is applied on the shaved skin area, which acts asa lubricant for the metal microarray needle. AdminStamp 600 MicroneedleArray Device is used to create micropores in the skin tissue. Thisdevice has 187 five hundred micron tall stainless steel microneedles on1 square centimeter (circular shape). The array is inserted in thetissue, rotated 180 degrees for 3 times and removed. The sterile PLGAsolution is applied on the skin and a sterile nitrogen get pressurizedstream is applied on the solution. The solution is allowed to incubatewith the porous area for 10 minutes. The excess solution from the tissueis wiped off using sterile gauze.

About 10 treated areas are treated in a similar fashion. The treatedtissue is cut and some tissue subjected to histological processing tosee the presence of deposited polymer in the tissue. The other tissue isused to monitor the release of moxifloxacin from the tissue. Briefly thecut tissue is incubated in 3 ml PBS and drug release is monitoredseveral times for 30 days. PBS is changed every time when drug solutionsample is collected to maintain sync conditions.

Creating porosity and filling injectable composition at the same time.

Applying the injectable composition (polymer solution) first as afluid/liquid layer and then applying microneedle array or oscillatingneedle to form pores and fill the cavity.

Bovine pericardium tissue (5 cm by 5 cm) is procured freshly and isdecellularized. The tissue is incubated for 24 hours in PBS to hydrateit completely. The pericardial tissue can be considered as exemplarysurgical bioprosthesis patch or wound dressing. The sterile moxifloxacinsolution from part 1 as above is first applied on the tissue (about 2square centimeter area, (about 0.1 to 1 mm solution layer thickness) andthe sterile AdminStamp 600 Microneedle Array Device is applied on thesolution and pressed into tissue. The needles penetrate the tissue andits needle carry solution with it inside the tissue. The solution istransferred by the microneedles inside the tissue. The needles areremoved from the tissue and reinserted in the same area. This isrepeated 2 or more times. The excess solution is wiped off. About 10areas are treated this way. The treated tissue is incubated in PBS for 2h to accelerate precipitation of the polymer inside pores. The polymerin the tissue precipitates entrapping the drug inside the precipitatedPLGA. Methylene blue provides blue color, which helps to visualize thetreatment. The infused portion is clearly visible on the white tissuebackground. The infused section is observed under scanning electronmicroscope and regular microscope to confirm the presence of polymermicroimplants. The treated tissue section is cut from the tissue andsent for histology analysis to confirm the formation of PLGAmicroimplants at the treatment site. In another experiment the infusedsection is cut from the tissue is incubated in 3 ml PBS at 37 degree C.Fresh 3 ml PBS is exchanged at following time intervals: 30 minutes, 60minutes, 12 h, 24 h, 2 day, 3 day, 5 day, 7 day, 14 day, 28 day timeperiod. The drug eluted sample are protected from light and stored inrefrigerator until HPLC or UV spectrophotometer analysis. The elutedmoxifloxacin in PBS solution is analyzed using UV spectrophotometer.

In another modification of this embodiment, 20 ml dimethyl sulfoxide,1.4 g Poly (PLGA, lactide-co-glycolide) (dl-lactide:glycolide (75:25),molecular weight 30000 to 60000 g/mole.) and 600 mg (approx. 30 percentloading relative to weight of the polymer plus drug) bupivacaine base isused and the solution is infused in pericardial tissue using 20 needlearray as shown in FIG. 22A. The infused drug is eluted in PBS asmentioned above and is analyzed using UV visible spectrophotometer orHPLC.

In another modification of this embodiment, 20 ml dimethyl sulfoxide,1.6 g polycaprolactone (molecular weight 70,000-90,000 g/mole.) and 400mg (approx. 20 percent loading relative to weight of the polymer plusdrug) rifampin is used and the solution is infused hollow microneedle 3by 3 microneedle array (33 MP). The infused drug is eluted in PBS asmentioned above and is analyzed using UV visible spectrophotometer.

In another modification of above examples, gentamycin is replaced withrifampin, or chlorhexidine diacetate salt hydrate. In anothermodification, moxifloxacin in the above examples is replaced withcoumarin 6 a fluorescent visualization agent and model drug.

Example 10B

Formation of Artificial Porosity Using Oscillating Needle and Depositingthe Polymer Solution in the Cavity.

Formation of In Situ Biodegradable Microimplant Array in the TissueUsing Oscillating Needle Method.

Part 1: Synthesis of Polyethylene Oxide (PEO)-polypropylene Oxide(PPO)-polyethylene Oxide Lactate Copolymer (PEO-PPO-PEO LactateCopolymer)

20 g of Pluronic F127 (PEO-PPO-PEO block copolymer) is dried undervacuum at 100 degree C. for 24 h. 20 g of dry Pluronic F127, 4.61 g ofdl-lactide and 30 mg of stannous octoate are charged into 100 ml Pyrexpressure sealing tube. The tube is then connected to argon gas line andsealed under argon. The tube is then immersed in oil bath maintained at140 degree C. and the reaction is carried out for 16 h at 140 degree C.The polymer from the tube is recovered by breaking the Pyrex tube. Thepolymer is then dissolved in 100 ml chloroform and precipitated in 2000ml cold hexane or ether. The precipitated polymer is recovered byfiltration and dried under vacuum for 1 day at 60 Degrees C.

Part 2: Injection of PEG Copolymer (PEO-PPO-PEO Lactate Copolymer) inthe Prosthetic Tissue Using Microarray

In a 50 ml beaker, 20 ml n-methyl pyrrolidone, 1.8 g PEO-PPO-PEO lactatecopolymer and 200 mg (approx. 10 percent loading relative to weight ofthe polymer plus drug) rifampin are added until solution is formed. Thesolution is sterile filtered using PTFE based filter in a clean sterilepolypropylene tube. The solution is first applied on the tissue surfaceto form a 100-900 micron thick liquid layer on the surface. The tattoomachine needle is used to insert the solution on the tissue to form acavity as well as to insert the solution in the cavity. The needle goesin and out of the tissue at 10-12000 minutes per minute. The solutionspreads on the needle surface and penetrates in the tissue and depositsin the tissue where needle has inserted. The deposited solutionprecipitates in the tissue cavity and form drug delivery microimplant insitu. Total one centimeter square area of the tissue is treated for 1minute. The infused drug is eluted in PBS as mentioned above and isanalyzed using UV visible spectrophotometer. The needle penetration areais chosen in such way that a microimplant array is formed. In thismethod, no microneedle array is used to create a microimplant array.

In another modification of above example in Part 1, 20 g of polyethyleneglycol (molecular weight 20000) g/mole is reacted with 14.4 g lactideand 30 mg stannous octoate are reacted at 140 degree C. for 16 h toproduce PEG-polylactide high molecular weight polymer (molecular weight30000 to 40000 g/mole). A 10 percent of this polymer solution in acetoneis used for infusion with tattoo machine.

In another embodiment of above example in Part 1, 2.00 g polyethyleneglycol (molecular weight 2000 g/mole), 7.2 g of dl-lactide, 5.7 gcaprolactone and 30 mg of stannous octoate are reacted at 140 degree C.for 16 h to produce PEG-co-polylactide-co-polycaprolactone copolymer. A10 percent of this polymer solution in DMSO is used for infusion usingoscillating needle in part 2.

The molar ratio of cyclic lactone and hydroxy groups in the PEG orPluronic polymers is used to control the molecular weight (degree ofpolymerization) in the copolymer. The PEG-polylactone ratio may bechanged 5-90 percent to obtain polymers with wide range of propertiesincluding thermoreversible properties, water solubility, solid/liquidnature at room temperature and the like. Some of PEG-polylactonepolymers are water soluble and some of them are water insoluble.

In another modification of above examples, rifampin is replaced withgentamycin, chlorhexidine diacetate salt hydrate. In anothermodification, rifampin in the above examples is replaced with coumarin 6as a fluorescent additive and model drug.

Example 10C

Delivery of Injectable Synthetic Biodegradable Polymer Solution in theArtificial Porosity.

Delivery of Water Soluble Synthetic Biodegradable Polymer.

Part 1: Synthesis of Water Soluble Polyethylene Glycol Lactate Copolymer(PEG-polylactate-10)

In a 500 ml flask, 20.0 g of PEG 10000 (molecular weight 10000 g/mole),and 200 ml toluene is added. Approximately 80-100 ml toluene isdistilled of and the solution is cooled. 5.4 g of dl-lactide and 30 mgof stannous octoate are added in the flask and the solution is refluxedfor 24 h under nitrogen atmosphere. The flask is cooled and the solutionis precipitated in 2000 ml cold hexane or ether. The precipitatedpolymer (PEG-LACTATE-10) is recovered by filtration and dried undervacuum for 1 day at 60 degree C.

Part 2: Injection of PEG Copolymer Lactate Copolymer(PEG-Polylactate-10) in the Tissue Using in the Artificial Porosity.

In a 50 ml beaker, 6 ml PBS, 2.0 g (PEG-polylactate-10) and 100 mg(approx. 5 percent loading relative to weight of the polymer plus drug)rifampin are added until complete solution. The solution is sterilefiltered using PTFE based filter in a clean sterile polypropylene tube.1 ml of solution is poured on 2 cm by 2 cm sheep dermal tissue and thesolution is infused by inserting AdminStamp 600 microneedle device or 3by 3 hollow microneedle array from Micropoint Technologies (33 MP). Asurgical sealant such as Fibrin sealant or DuraSeal is applied on top oftreated area or band aid type adhesive tape or silicone rubber basedwound dressing is applied to prevent the injected solution to come outof cavities. The concentration of polymer in PBS is above its criticalmicelle concentration and therefore it forms micelles in PBS, which canentrap hydrophobic drugs. The drugs in the micelles are released in asustained manner. This example can be treated as micellar drug deliverysystem wherein drug is incorporated in the micelles and the micelles arethen injected artificial pores of the tissue. Each micelle can beconsidered as nano size drug loaded microparticle. In anothermodification, rifampin in the above examples is replaced with coumarin 6as a fluorescent additive and model drug.

Example 10D

Delivery of In Situ Forming Crosslinkable Compositions MicroarrayDevice.

Delivery of Composition that Crosslink Using Free Radical Polymerization

Part 1: Synthesis of Polyethylene Glycol Lactate Copolymer

In a 500 ml flask, 20.0 g of PEG 10000 (molecular weight 10000 g/mole),and 200 ml toluene is added. Approximately 80-100 ml toluene isdistilled of and the solution is cooled. 2.68 g of dl-lactide and 30 mgof stannous octoate are added in the flask and the solution is refluxedfor 24 h under nitrogen atmosphere. The flask is cooled and the solutionis and precipitated in 2000 ml cold hexane or ether. The precipitatedpolymer (PEG-LACTATE-5) is recovered by filtration and dried undervacuum for 1 day at 60 degree C. It then immediately used in nextreaction.

Part 2: End-Capping of PEG-LACTATE-5 with Polymerizable or CrosslinkableGroup (PEG-LACTATE-5-acrylate)

In a 500 ml reaction flask, 20 g of PEG-LACTATE-5 is dissolved in 300 mldry toluene. About 50 ml of toluene is distilled out to remove traces ofwater from the reaction mixture. The warm solution is cooled to roomtemperature. 0.39 g of triethyl amine and 0.34 g acryloyl chloride areadded. The reaction mixture is then stirred for 6 h at 50-60 degree C.and filtered. PEG-LACTATE-5-acrylate macromonomer is precipitated byadding the filtrate to 2000 ml cold hexane or ether. The precipitatedpolymer is recovered by filtration. It is then dried under vacuum for 12h at 50 degree C.

Part 3: Polymerization and Crosslinking of Deposited Solutions in theArtificial Cavities.

Separately 3 g of PEG-LACTATE-5-acrylate diacrylate prepared as above isdissolved in 9 g PBS. 300 mg Irgacure 2959 is dissolved in 700 mgn-methyl pyrrolidone. 50 microliter of Irgacure 2959 solution is addedto the PEG-LACTATE-5-acrylate solution and 100 mg heparin as model watersoluble drug is added to the solution. The solution is sterile filteredusing 0.2 micron filter. The sterile solution (precursor solution) isfilled inside a sterile syringe and the syringe is attached to sterile 3by 3 stainless steel hollow microneedle hub from Micropoint Technologies(33 MP). The microarray is inserted inside the sheep skin tissuecompletely. The needle is pulled approximately 80 percent out and thespace created during pulling is filled with the sterile precursor. Theinfused solution used then exposed to the long UV ultraviolet light(Black-Ray UV lamp, 360 nm light, 10000 mW/cm2 intensity) for 5 minutesto photopolymerize and crosslink the infused precursor solution in thetissue. The PEG-LACTATE-5-acrylate polymerizes and crosslinks to formbiodegradable hydrogel particles inside the tissue. This leads toformation 3 by 3 array of microimplants in the tissue. The process isrepeated at 10 locations on the tissue to increase amount of implantmaterial formed in the tissue. The entrapped crosslinked hydrogelrelease the drug in a sustained manner.

In another modification as above, 30 g PEG 10000 (tetrafunctional, oneterminal hydroxy group per PEG branch, total four branches) is reactedwith 8.460 g dl lactide in first part and 1.668 g acryloyl chloride and1.882 g triethyl amine in second part. The macromonomer formed has fouracrylate groups per molecule which upon polymerization and crosslinkingproduce crosslinked hydrogels that have higher crosslinking density thanits bifunctional counterpart produced as above. In another modificationas above, 30 g PEG 20000 is reacted with 2.130 g dl lactide in firstpart and 0.501 g acryloyl chloride and 0.565 g triethyl amine in secondpart to produce PEG 20K-lactate-acrylate macromonomer.

In another modification as above, 30 g PEG 35000 is reacted with 1.234 gdl lactide in first part and 0.295 g acryloyl chloride and 0.333 gtriethyl amine in second part to produce PEG 35K-lactate-acrylatemacromonomer.

By changing PEG molecular weight, number of acrylate groups per PEG andpolylactones as indicated above, crosslinked hydrogel networks withvariety of degradation times and molecular permeability can be made andcan be used in variety of drug delivery and cell encapsulationapplications. In case of islet encapsulation, molecular permeability isadjusted by variables mentioned as above so that molecules with 100000g/mol molecular weight can go in and out of the islet encapsulatedhydrogels matrix, but will not allow to permeate/diffuse immunoglobulins(IgG, molecular weight around 150000 g/mole) to diffuse through thecrosslinked hydrogels.

In another experiment, a 500 micron thick spacer (500 micron thickpaper, Teflon or polyester film is placed between needle and hub whereneedles are attached. The spacer limits the depth of penetration from1000 microns to 500 microns. Alternatively, a 500 microns tall needlearray specifically fabricated for this purpose and used. Other spacersof various thickness (50, 100, 900 microns and the like) may be appliedto limit the depth of penetration of needle in the tissue and hencedepth of artificially cavity.

In another modification as above, a visible light photopolymerization isused to crosslink the injected precursor solution in the cavities. In100 ml beaker 3 g of PEG-LACTATE-5-acrylate diacrylate prepared as aboveis dissolved in 9 g PBS. In another 10 ml glass vial, 300 mg eosin Y isdissolved in 700 mg n-vinyl pyrrolidinone. 30 microliter of eosin Ysolution, 1 ml of 5 M triethanol amine in PBS are added toPEG-LACTATE-5-acrylate solution and the solution sterile filtered andprotected from light using aluminum foil. The precursor solution isinfused in the pericardial tissue or porcine dermal tissue using atattoo machine like oscillating needle at a depth of 10-1000 microns orinserted in to 3 by 3 cavities created by 33 MP array. The infusedsolution is crosslinked by photopolymerization by exposing it to the 512nm laser (argon laser) light or high intensity white floodlight. Thelight polymerizes and crosslinks the PEG-LACTATE-5-acrylate monomer andforms gel particles in situ inside the cavity. In another modificationof same example, about 1 million fibroblast cell suspension (1 millioncells suspended in 0.2 ml of are added to 1 ml of sterile filteredprecursor solution prior to injecting in the tissue in the dermal tissueor live tissue. The injected solution is crosslinked using visible light(512 nm Laser). The 512 nm light can penetrate upto 1000-3000 micron dipin the tissue and is therefore is capable of polymerization andcrosslinking the precursor formulation without damaging the cells. Thepolymerized cells in the crosslinked materials may be used in cell basedtherapy.

In another embodiment, 200 mg of PEG 35K-lactate-acrylate macromonomerprepared as above is dissolved in 800 mg PBS. After completedissolution, 200 mg of magnesium carbonate is added as opacity creationagent or as visualization agent or as a filler. 300 mg Irgacure 2959 isdissolved in 700 mg n-methyl pyrrolidone. 5 microliters of Irgacure 2959solution is added to the macromonomer solution. The sterile solution(precursor solution) is filled in the array of cavities (4 by 4 array)created in sheep tissue, excess solution is wiped off and exposed tolong UV ultraviolet light (Black-Ray UV lamp, 360 nm light, 10000 mW/cm2intensity) for 5 minutes to photopolymerize and crosslink themacromonomer solution to form crosslinked hydrogel. FIG. 19A showscrosslinked biodegradable hydrogel 4 by 4 microimplant array in sheeptissue. The crosslinked hydrogel with magnesium carbonate asvisualization agent or filler (1901) is clearly seen in the image. Inanother modification of above embodiment, 100 mg tissue plasminogenactivator (TPA, an exemplary protein drug) is added in place of cells.The TPA is entrapped in a hydrogel particle and is then released in asustained manner when the crosslinked PEG-LACTATE-5-acrylate degrades invivo.

In another modification, inorganic biocompatible, biodegradable fillerssuch as magnesium carbonate, calcium sulfate, and the like may be mixedwith crosslinkable polymer solution such as PEG 35K-lactate-acrylatemacromonomer solution as above and polymerized and crosslinked withlight to entrap the filler in the gel. The entrapped filler can be usedto alter the local chemical/physical environment of the crosslinked gel(pH, osmolality, surface area and the like) which may help to tune thesustained drug release of drugs.

Example 10E

Microarray Made Using In Situ Crosslinkable Compositions

Composition that Crosslink Using Condensation Polymerization

500 mg PEG10KARM glutarate NHS ester obtained from commercial sources;(molecular weight 10000 g/mole, 4 arm star shaped, with terminal NHSgroups and glutarate as degradable ester, Laysan Bio Inc., Arab, Ala.)is dissolved in 9.5 ml PBS (20 mM pH 7.2) until complete dissolution(precursor A solution). 2 g albumin and 10 mg methylene blue isdissolved in 9 ml PBS (precursor B solution). Both precursor solutionsare sterile filtered. 1 ml of PEG10KARM glutarate NHS ester solution and1 ml albumin are loaded in duel syringe [(Duel syringe Product code6B23-3 ml×3 ml, 1:1 Ratio, with 2 mm×8 Element mixer tip) from Pals-PakIndustries, Inc., Norwich, Conn., USA] is used. The output of duelsyringe is fed to 3 by 3 hollow microarray from Micropoint Technologies,33 MP. The gel time for this solution is about 20-180 seconds. The arrayis inserted into sheep dermal tissue and the precursors solutions arepushed from both the syringes, which are mixed and infused into tissuecavities created by the array. The array is removed from the surfaceleaving behind empty space filled with crosslinkable composition. Theinfused precursor mixture undergoes condensation polymerization andcrosslinking (total reactive functional groups in the precursors must begreater than 5 and each precursor must have greater than 2 functionalgroups). The precursors react with each other forming gel particles atthe injection site. If the precursors are loaded with drug, the drug isentrapped in the crosslinked gel and is released in a sustained manner.The drug should not have functional group capable of reacting withprecursors under crosslinking conditions.

In another embodiment, PEG10KARM glutarate NHS ester and trilysine aremixed in molar equivalent quantities. The gel time of the precursors areadjusted using various buffers that provide pH in 6 to 8 range such asPBS buffer with PH 7.8 or sodium acetate buffer with 6 and the like). Ingeneral, acidic pH is preferred. Some of the formulations gels in fewseconds and therefore may be preferentially used by mixing in situinside the duel syringe device before injecting in the tissue via 3 by 3microneedle array.

Another modification of above example, albumin is replaced with gelatinor collagen solution (1-5 percent in PBS or 0.1 M acetic acid) to form acrosslinked gelatin or collagen gels.

Another modification of above example, albumin is replaced with PEG10000 tetrafunctional amine terminated polymer (PEG10K-4Amine, molecularweight 10000 g/mole, 4 arm, terminal amine groups, available from LaysanBio, Inc. AL). Molar equivalent quantities of PEG10KARM glutarate NHSester and PEG10K-4Amine are mixed in PBS pH 7.8 and injected in tissueusing 33 MP array. The solutions are allowed to react in the cavity for10 seconds to 300 seconds. The crosslinked gel array is used for drugdelivery. In another modification of same embodiment, rifampinmicrospheres made in Example 3 are mixed with PEG amine and then reactedin situ in the cavities to form a gel the PLGA microspheres embedded inthe gel. The rifampin is released from the gel in a sustained manner.

Example 10F

Delivery of In Situ Forming Ccrosslinkable Compositions UsingOscillating Needle Device or Hollow Microneedle Array.

Delivery of composition that crosslink via enzymatic pathway.

Formation of fibrin gels particles in situ using cavity fillingtechnique.

A commercially available EVICEL® from Ethicon or TISSEEL from Baxter maybe used. The components of fibrin glue (fibrinogen, thrombin, factor 8,calcium ions and the like) are supplied as a two component mixture. Thecomponents of commercially available as fibrin sealant and are mixed ina sterile cup (total volume of mixed components 1-2 ml). To thissolution 5 drops ophthalmic sodium fluorscein solution are added or 10mg of indocyanine green dye added as a fluorescent/coloring agent. If nocolor is desired, the formulation can be used without the use ofcoloring agent or dye. The colored fibrin formulation is then loaded ina sterile syringe and it is connected in the 3 by 3 stainless steelhollow microneedle hub from Micropoint Technologies and injected intopericardial tissue or in live peritoneal tissue. The excess solution onthe tissue surface is wiped off. The injected solution in the 3 by 3array cavities undergo enzymatic polymerization/crosslinking and formfibrin glue/gel particles in situ inside the artificial cavities createdinside the tissue. If desired, the components of fibrin glue aredelivered via a duel syringe apparatus and then connected to hollowneedle microarray hub. The precursor components are mixed in the mixingchamber and then delivered to microarray needles. Care is taken toinject the formulation before the fibrin glue forms gel (usually 1-2minutes). If components prematurely gel, then a new mixture is preparedand used quickly before gelling. The fibrinogen solution may be dilutedusing PBS to slow the gelation process. Alternatively, a modified duelsyringe based device [(Duel syringe Product code 6B23-3 ml×3 ml, 1:1Ratio, with 2 mm×8 Element mixer tip) from Pals-Pak Industries, Inc.,Norwich, Conn., USA] may be used wherein the components are mixed insidethe device just prior to injection and injected by the hollow microarrayneedles. The colorant or fluorescence of particles or droplets providesvisual clue on the amount of injected solution at each injection site. Adrug may be added to the composition. Drugs that interfere with thefibrin glue formation such as TPA or heparin cannot be used for localdelivery using this method. Many drugs can be used with fibrin gluesystem. Live cell suspensions may be added to the fibrin glue componentsto deliver live cell based compositions. A multilumen needle may be usedto deliver fibrin glue precursors (one lumen for fibrinogen solution)and another lumen for thrombin solution.

The components are injected simultaneously and crosslinked in situ.

Example 10G

In situ formation of water insoluble drug solids in the artificialporosity.

In situ microimplants made using in situ precipitated drug crystals.

In a 50 ml glass baker, 1 g of chlorhexidine diacetate salt hydrate and10 mg ethyl eosin or methylene blue as a colorant are dissolved in 20 mlethanol. The solution is sterile filtered. The solution is deposited inthe skin tissue using 3 by 3 hollow microarray from MicropointTechnologies as described previously. Upon deposition in the tissue andunder physiological conditions (37 degree C., pH 7.4), the ethanol inthe solution is dispersed in the tissue and leaving behind substantiallywater insoluble chlorhexidine diacetate as solid crystals at theinjection site or in the artificial cavity space created by the array.

In another variation of above embodiment, 10 mg of paclitaxel, ananticancer drug is dissolved in 10 ml dimethyl sulfoxide or ethanolalong with 10 mg of methylene blue or ethyl eosin or turmeric as acolorant. The DMSO solution is injected in using hollow array (33 MP) asdescribed above leaving behind paclitaxel crystals upon dissipation ofDMSO by the tissue. This creates a 3 by 3 array of cavities filled withpaclitaxel crystals which dissolves slowly for local therapeutic effect.

Example 10H

Delivery of Thermosensitive Compositions in the Artificial Cavities.

Preparation of PEG Based Thermosensitive Polymer

In 250 ml connected to a condenser and nitrogen inlet, 2.55 g (propyleneglycol)-block-poly(ethylene glycol)-block-poly(propylene glycol)bis(2-aminopropyl ether) (Jeffamine® XTJ-502, Molecular weight 1900g/mole, Aldrich) is dissolved in 100 ml toluene. About 30 ml toluene isdistilled off and cooled. 5 g dl-lactide (Aldrich) and 0.2 ml stannousoctoate is added and the mixture is refluxed for 4 hours under nitrogenatmosphere. The mixture is cooled and poured into cold hexane toprecipitate the Jeffamine-lactide copolymer. The product is isolated byand dried under vacuum and stored in desiccator until use. The A 20-30percent of Jeffamine lactide solution in PBS shows thermoreversiblegelation around 30 to 40 degree C. 1 ml of 30 percent of Jeffaminelactide solution is mixed with 200 mg of rifampin microencapsulatedmicrospheres (Example 3) and the suspension is infused into 4 by 4microarray micro-cavities created manually on a sheep skin tissue. FIG.22D shows the sheep skin tissue with 4 by 4 microarray implantcontaining Jeffamine lactide copolymer thermosensitive gel (an exemplarythermosensitive gel array) and rifampin encapsulated microspheresentrapped in the gel (red colored 2207).

Preparation of Pluronic Based Thermosensitive Polymer

In a 250 mL glass beaker, 20 g of Pluronic F127, 0.5 g of chlorhexidineacetate or chlorhexidine gluconate and 10 mg of methylene blue aredissolved is 40 g cold PBS solution (0-10 degree C.). The F127 is aPEO-PPO-PEO block copolymer that has thermoreversible gelationproperties. The cold polymer solution is sterile filtered (temperatureheld at 0-10 degree C. during filtration). The cold solution is used fordeposition in the artificial porosity as described before. The solutionis filled in the syringe and kept cold using an ice bath to maintain itsfluid state. The solution is deposited using 3 by 3 hollow needle arrayfrom Micropoint Technologies (33 MP). Briefly, syringe with coldsolution is attached to 3 by 3 array hub (33 MP) which is precooled toaround zero degree C. in the ice bath. The array is then inserted in theporcine dermal skin or Alumax™ Surgical Graft (size 16 by 20 cm size)from C. R. Bard Inc. and the cold solution is injected in the tissue.The tissue is then incubated at 37 degree C. for 5 to 30 minutes toconvert the cold solution into Pluronic gel in the injected space. Thegel formed releases the drug in a sustained manner. In anothermodification of above example, the cold Pluronic solution is applied onice cold tissue to form a liquid layer on the tissue. A 33 MP array asabove or AdminStamp 600 Microneedle Array is pressed on cold solutionthrough the solution to form micro cavities in the tissue as well as todrive the solution inside the formed cavities. The tissue is warmed to37 degree C. and the liquid in the cavities undergo thermoreversiblegelation and form a gel inside the cavities. The same experiment can becarried on live human on porcine skin tissue. The cold solution isapplied on the skin tissue and is forced into cavities using themicroarray device. Due to warm body temperature of live pig or humanskin, the Pluronic solution is converted into Pluronic gel and staysinside the cavities as a gel. If drug is loaded in the Pluronic gel, itis released in the cavity in a sustained manner. Care is taken so thatthe Pluronic solution stays in liquid state and not in gel state duringapplication. If it forms a gel on the skin due to warm body temperature,then it may be cooled to less than 10 degree C. to using ice pack toliquefy the gel to a solution and forced into cavity while in liquidstate.

In another modification of the above example, a thermoreversible polymerthat forms solution when warmed around 40-65 degree C. but forms gelwhen cooled to body temperature or room temperature is used. A gelatingrade that is soluble in hot water but not in cold or body temperaturewater is used. Briefly 10 g of gelatin, 90 g PBS and 10 mg indocyaninegreen as a green colorant is used. The hot gelatin solution (40-60degree C.) of gelatin is deposited inside the tissue using hollowmicroneedle array as above.

Some PEG-polylactone polymers (known in the prior art) also showthermoreversible gelation similar to gelatin or Pluronic 127 and suchpolymers may also be used to deposit in the tissue as described above.Variables such as concentration of polymer in the solution, geltransition temperature and the like may be determined experimentally orcan be found using polymer chemistry literature.

Example 10I

Delivery of Low Melting Compositions in the Artificial Porosity.

Delivery of Compositions that Form Microarray In Situ

In a 250 mL glass beaker, 20 g of Pluronic F127 and 0.1 g ofchlorhexidine acetate or gluconate and 10 mg of ethyl eosin are addedand mixed. The mixture is heated to 60 degree C. in an oil bath untilF127 polymer melts. The melted polymer is mixed thoroughly withchlorhexidine diacetate salt hydrate and cooled and pulverized usingmortar pastel. The melted drug polymer composition is re-melted in anoil bath maintained at 60 degree C. and used for deposition in theartificial cavities of bioprosthesis tissue or in the live tissue.Briefly, about 3 g of Pluronic F127 and chlorhexidine composition asabove transferred in the syringe. The syringe and 3 by 3 microarray hubfrom Micropoint Technologies (33 MP) are preheated to 60 degree C. Thepolymer is allowed to melt completely. The hot polymer melt forms a lowviscosity liquid at 60 degree C. The hot array hub is connected to thesyringe via Luer Lock connector on the hub. The array is completelyinserted in the porcine dermal tissue and pulled back about 90 percentof the needle length. The cavity created by pulling back is filled bypushing the syringe and pushing the melted polymer in the cavities. Theexcess melted polymer on surface is wiped off. If needed, band aid orsurgical sealant or fibrin glue or cyanoacrylate glue and the like maybe applied on treated surface to prevent migration of implant from theinjected space. The polymer is allowed to cool to room temperature or tobody temperature. The cold polymer solidifies in the injected artificialcavity and entraps the drug. The drug is released from the cooled solidin a sustained manner. If necessary, hot air may be blown using a hairdryer on the array hub and or syringe to prevent the prematuresolidification of the composition in the hub or apparatus. In anothermodification of above example, Pluronic F127 is replaced with lowermolecular weight Pluronic F68. In another modification, chlorhexidineacetate is replaced with rifampin or coumarin 6.

In another modification of the above example, polycaprolactone polymer(molecular weight 2000 g/mole) is used for drug delivery and depositionusing microneedle hub as described above. Briefly, in a 100 ml beaker,9.9 g polycaprolactone polymer, 0.1 g rifampin and 10 ml dichlormethaneare added until complete homogenous solution (drug weight percent is 1percent relative to polymer plus drug weight). The methylene chloride isremoved by air drying inside the hood leaving behind the polymer anddrug. The polymer is vacuum dried over night at 40 degree C. The polymeris heated in oil bath to melt the polycaprolactone at 50-60 degree C.The liquid polymer is then filled inside the syringe and depositedinside the pericardial tissue using the hollow microneedle hub asdescribed above. The excess polymer on the tissue surface is wiped off.The deposited liquid polymer cools and forms solid microparticles insideporous cavities created by the hub.

In another modification of the above embodiment, a bone wax is used inplace of polycaprolactone. The wax melts around 60 degree C. and can beinjected and cooled to form wax particles. The copmposition is abiostable non-polymer or oligomeric low melting composition. In anothermodification of the above embodiment, a D-a-Tocopherol polyethyleneglycol 1000 succinate is used as low melting polymer is used in place ofpolycaprolactone.

In another modification of the above embodiment, steric acid is used asa non-polymeric solid in place of polycaprolactone.

In another modification of the above embodiment, a low melting (below 60degree C.) PEG-polylactone or PEG-polycaprolactone polymer is used inplace of polycaprolactone.

Example 10J

Delivery of Biocompatible Liquid Compositions in the Artificial Porosityin the Tissue.

Liquid Sustained Drug Delivery Arrays Formed In Situ.

In a 250 mL glass beaker, 20 g vitamin E acetate and 1 g of rifampin areadded. The non-polymeric liquid carrier (vitamin E acetate) is thenfilled in the artificial cavity space or arrays created in the tissue.Briefly, 3 ml of vitamin E acetate with rifampin as above is transferredin disposable syringe without needle with male Luer lock. A 3 by 3hollow microneedle array from Micropoint Technologies (33 MP) withfemale Luer lock is attached to the syringe. The array is then insertedin the porcine dermal tissue. It is pulled back 80 percent of the needlelength and the vitamin liquid is pushed from the syringe via array inthe tissue cavity created by pulling back the needle array. The liquidis deposited and excess liquid is pushed off from the array. The arrayis removed and the excess liquid on the tissue is wiped off. The liquidfilled cavities in the injected areas release the drug from liquidcarrier Vitamin E acetate. If desired a band aid type layer may beapplied on the treated area or tissue sealant like fibrin glue (TISSEEL®or EVICEL®) or DuraSeal® surgical sealant available commercially. Thesealant may prevent unnecessary movement of the liquid microimplantarray during routine physical activity like walking, running and thelike.

In another embodiment, 1 g of vitamin E acetate is mixed with 100 mg ofmagnesium carbonate stained with tea stain. The dark colored suspensionis used to fill cavities of 10 by 10 array created in sheep tissue. FIG.19B shows a picture of liquid (vitamin E acetate) microimplant arraywith red colored liquid microimplants arranged in 10 by 10 array format.The stained magnesium carbonate is added as a biocompatiblebiodegradable visualization agent or filler.

In another modification of the above example, a biodegradable polymericliquid (polycaprolactone polymer; molecular weight 520 g/mole) is usedfor drug delivery and deposition using hollow needle array. The polymeris liquid at ambient or body temperature. Briefly, in a 100 ml beaker 9g polycaprolactone polymer and 100 mg rifampin are added until completehomogenous solution/suspension is formed (drug weight percent is 10relative to polymer plus drug weight drug weight). The liquid polymer isthen filled inside the artificial cavities as described above (usinghollow microneedle array 33 MP) or in this invention. The excess polymerliquid is wiped off from the tissue surface. The deposited liquidpolymer delivers the drug in a sustained manner. The liquid polymer isremoved from the tissue by the biodegradation process.

In another modification of the above embodiment, sucrose acetateisobutyrate solution in ethanol or NMP, a non-polymeric liquid is usedin place of polycaprolactone. The solvent NMP or ethanol is added tomodify the viscosity of the sucrose acetate isobutyrate. Only smallamount (1-20 percent) solvent is added to make it suitable for injectionusing the array 33 MP. The solvents used (DMSO, ethanol, NMP and thelike) preferably are water miscible, biocompatible and biodegradable.

In a 500 ml flask, 18.0 g of PEG 1000 (molecular weight 1000 g/mole),and 200 ml toluene is added. Approximately 80-100 ml toluene isdistilled of and the solution is cooled. 2 g of dl-lactide and 30 mg ofstannous octoate are added in the flask and the solution is refluxed for24 h under nitrogen atmosphere. The flask is cooled and the solution isand precipitated in 2000 ml cold hexane or ether. The precipitatedliquid polymer (PEG1000-LACTATE) is recovered by decanting the solventand drying under vacuum for 24 h. The PEG molecular weight and molarratio or PEG to lactone (l-lactide, dl-lactide, glycolide, caprolactoneor trimethylene carbonate) is varied to obtain liquid (liquid at 37-40degree C.) PEG-polylactone polymers of various viscosities anddegradation times. The polymers can be used as liquid carriers asmentioned previously.

In another modification of the above embodiment, oleic acid, anon-polymeric liquid is used in place of polycaprolactone. This carrieralso could also be used with water miscible solvents as described aboveto adjust its viscosity.

In another embodiment as above, rifampin is replaced with coumarin 6 asmodel hydrophobic and fluorescent drug and colorant.

Example 11

General method for in situ array formulation development.

Porosity Creation

-   -   Porosity creation using microneedle array:    -   Microneedle Array Device Variables: Device material (metal,        polymeric, proteins, natural macromolecules, glass, silicon,        ceramic, ice, sugars and the like), microneedle length, needle        angle while tissue penetration, microneedle shape, microneedle        edge shape, dissolvable or inert, number of needles, distance        between needles in the array, needle volume, needle porosity        (hollow or solid); microneedle backing material, array shape,        array area size, density of array (needles per centimeter square        or area).        Laser Induced Porosity Variables:    -   Wavelength (ultraviolet or visible or infrared), laser spot        size, laser power, laser pulse frequency, pulse repetition        rates, number of stacked pulses.    -   Oscillating needle based variables:    -   Needle size and shape, oscillation frequency, needle edge,        number of needles, needle materials (metal, ceramic or        polymeric), tissue penetration length;        Mechanical Drilling Based Variables:    -   Drill bit size (diameter), drill bit material type, drill        rotation speed, drill penetration depth.

Injectable Composition Variable: Type of polymer, polymer molecularweight, polymer concentration in the solvent, type of solvent, solventconcentration, viscosity of composition, drug concentration, wettingagents type and amount added, microparticle size, microparticle shapeand microparticle porosity, microparticle color, crosslinking chemistry,crosslinking type, precursor concentration and the like.

Drug type (synthetic or natural organic molecule, protein, syntheticpeptides), molecular weight, water and organic solvent solubility,partition coefficient, biological activity, stability in thecomposition, drug concertation in the composition, compatibility withnon-bioactive portion of the composition.

Array in Array Device Variables:

Base Array

-   -   Device material including base plate and needle(metal,        polymeric, proteins, natural macromolecules, glass, silicon,        ceramic, ice, and the like), hollow microneedle length, needle        angle while tissue penetration, microneedle shape, microneedle        distal edge shape and cutting angle, number of needles, distance        between needles in the array, needle cavity shape and volume,        lubricant on needle cavity surface, array shape, array area        size, density of array (needles per centimeter square), number        of guide posts and their size, protective cap for needles and        its material type, spacer lock shape, dimensions and material        type, force required to penetrate tissue and the like.        Plunger Array    -   Device material including base plate and needle(metal,        polymeric, proteins, natural macromolecules, glass, silicon,        ceramic, ice, and the like), solid microneedle length,        microneedle shape, microneedle distal edge shape and cutting        angle, number of needles, hollow passage shape and dimension,        injection port on proximal end (size and type), distance between        needles in the array, needle cavity shape and volume, lubricant        on needle wall surface, array shape, array area size, density of        array (needles per centimeter square or area), number of holes        for guide posts and their size, protective cap for needles and        its material type and the like.    -   Pre-formed microimplant in base array cavity or in implant        holding cartridge Implant size and shape, drug concertation,        number of live cells and cell type, number of live cells per        unit volume, unibody materials type (hydrogel, solid,        biodegradable, biostable and the like), porosity, porosity        filling material, visualization agent and its concentration,        other formulation specific non-drug additives like stabilizer,        antioxidant and their concentration, drug encapsulation matrix,        drug release rate from the implant, in vivo implant        biodegradation time, implant volume, surface area and the like.

Test Substrate: Pericardial tissue, transparent or semitransparentthermoreversible gel like gelatin gel (Jell-O like gel cast in a petridish), sheep or porcine skin tissue, tissue based hernia or surgicalpatch, chicken leg muscle, cow femur, freshly harvested bovine orporcine explanted organs like eyes, hearts, arteries, and the like.

Observations that can be recorded or made by changing various variablesas described above include but not limited to: The amount of compositioninjected per injection per needle, injection volume per injection perneedle, microimplant size, microimplant shape formed, implant depth ofpenetration, distribution and spacing of implant formed, drugconcentration in the implant, drug release rate in the injected area andthe like.

The injected composition in the tissue may be assessed by histologytechniques. Briefly the injected area of tissue may be cut and subjectedto histological techniques (encapsulating in wax or acrylic cement orfrozen tissue), drying, slicing, staining and observing microimplantsize by microscope or scanning electron microscope or electronmicroscope. A reconstruction of 3 dimensional distribution ofmicroimplant array size can be made from histological data.

The tissue with injected composition or gelatin gel may be dissolved in4 percent pepsin solution 0.1M HCL for 24 h at 37 degree C. to recoverimplants formed in situ. The recovered implants may be analyzed for sizeand distribution. Laser light scattering or SEM may be used to assessparticle size, distribution and shape. The gelatin gel orthermoreversible gels may be heated at around 60-70 degree C. or cooledbelow 10 degree C. to liquefy the gel. The implants in the liquefied gelmay be filtered and analyzed.

The injected tissue with microimplant array may be isolated andincubated in PBS under sink condition at 37 degree C. to eluteencapsulated drug of a period of time. The eluted drug may be analyzedby HPLC, UV-VIS spectrophotometer or other means. Rate of release (drugelution over a period of time) is then constructed from the observeddata.

A statistically designed experiment (DOE) may be used to test manyvariables discussed above, which can help to reduce the number ofexperiments needed to develop a suitable drug/cell delivery array asdescribed in this invention.

The list of variables and resulting data described above is a partiallist only and should not be considered as a limitation of thisinvention.

Example 12

Ophthalmic Drug Delivery Composition

In situ deposition of sustained drug delivery composition in corneal orscleral tissue Ten fresh bovine or porcine eyes are obtained from localslaughterhouse. 100 mg PLGA (50:50, molecular weight 8000-10000 g/moleor PDLG 5002 polymer), 5 mg coumarin 6 as fluorescent colorant ormoxifloxacin base a model hydrophobic drug and 0.9 ml polyethyleneglycol dimethyl ether are mixed in 15 ml glass vial. After completedissolution of all components, the solution is warmed to 37 degree toreduce its viscosity. 20 microliter of the solution is applied on thescleral tissue. AdminStamp 600 Microneedle Array Device with 187 fivehundred micron tall stainless steel microneedles or 33 MP array areapplied pressed on the tissue (needles facing scleral tissue) wheresolution is applied. The device is pressed until complete penetration ofneedles in the tissue. The device is rotated 180 degree while insertedin the tissue to form circular holes. The process is repeated (solution,application of needles, rotation and removal of needles from the tissue)two times on the same area. The excess solution wiped off. A 100microliter of PBS is applied 3 times to induce polymer precipitation insitu. The deposited solution precipitates in the scleral tissue andentrapped drug in the precipitated polymer is released in a sustainedmanner. In another example, another eye is used to deposit or injectdexamethasone loaded microspheres suspension in PBS (size less than 50microns, drug loading 20 percent, PLGA 50:50 polymer, molecular weight50000 g/mole suspended in PBS solution). The suspension is loaded in thesyringe. A 3 by 3 microarray hollow needle hub from MicropointTechnologies (33 MP) is attached to the syringe. The array is pressed onscleral tissue until complete penetration of needles in the tissue(needle length 300 microns by using 700 micron spacer on the needlehub). The array is pulled about 90 percent from the tissue and thesuspension is pushed form the syringe to fill the space created bypulling the needle. The array is removed and excess suspension is wipedoff. If needed, Resure™ ophthalmic sealant or fibrin sealant is appliedon the injection area to temporary lock the suspension inside thetissue. Tissues from untreated eyes and treated eyes are cut andanalyzed by histology. The drug elution from the cut section ismonitored by HPLC or UV-VIS spectrophotometer and a drug elution profileis generated.

In another embodiment, an array of cavities (4 cylindrical cavities in 2by 2 format, 200 micron dia, 200 microns height) are created using UVlaser or mechanical or other means in the scleral tissue (under theeyelid). The cavities are then filled using injectable compositions suchas PLGA solution with ophthalmic drugs like dexamethasone ormoxifloxacin. After implant formation via precipitation or crosslinkingand the like. If desired, the treatment area may be sealed using ReSure™ophthalmic sealant or fibrin sealant. Drug can be released from theimplanted array from few days to few months for many ophthalmicdiseases.

In another embodiment, prefabricated microimplants may be loaded in“array in array” device (Example 22). The base array device may beinserted up to 20 to 500 microns depth on ophthalmic tissue, preferablyin scleral or corneal tissue. The implants are pushed out using plungerarray device is withdrawn from the tissue. If desired, an ophthalmicsealant such as Resure ocular sealant may be applied to prevent leaksand infection. It is preferred that the implanted device does not breachtotal thickness of cornea or scleral tissue. The preferred implantoccupies/uses only 10-70 percent of corneal or scleral tissue thickness,preferably about 15 to 60 percent of thickness is used for implantationof ophthalmic implants. In one illustrative embodiment dexamethasoneloaded microimplant array as described above (total dose 0.4 mg perarray sustained released in 7-30 days) is used to treat postoperativepain and inflammation.

Example 13

In Situ Microarray Implants Made Using Dissolvable Arrays

Preparation of Degradable Polymer Implant

Dissolvable microarray implant is made using method as described inExample 6c or obtain commercially. A dissolvable array made usinghyaluronic acid is used to perforate sheep skin tissue. The array isinserted into skin tissue and allowed to dissolve. Some PBS solution maybe added to assist dissolution. After complete dissolution, a suctioncup and vacuum is used to aspirate biological fluids and dissolved arraymatter in the tissue. A polymer solution (PLGA solution (10 percent inNMP with rifampin (10 percent relative to polymer plus drug weight) isapplied on the treated area and allowed to permeate in the cavity.Exposure time 1 to 10 minutes. The solution fills the cavity spacecreated by dissolution of array. The excess solution is wiped off. Thedeposited solution in the cavities undergoes precipitation of polymer inthe cavity forming PLGA polymer entrapping the drug rifampin. In anotherexample, a fibrin glue components are dispensed from the duel syringeapplicator (provided by the fibrin glue manufacturer). The dispensefluid is spread on the array treated area and allowed to permeate insidethe artificial cavities. The excess solution is wiped of beforecrosslinking of fibrin components. The fibrin glue precursors depositedinside the cavity conform to the cavity shape and undergo gelation andform a fibrin clot in the cavity. If a protein such as bone growthfactor is added in the fibrin glue, it gets encapsulated in the clot andreleased from the clot.

Example 14

Degradable drug delivery microimplant array formed in situ and its drugrelease profiles Biodegradable polymer microimplant array formed in situusing microneedle array.

Example 14A

Array formed by direct injection of injectable composition via hollowmicroneedle array. Ten pieces of 2 cm by 2 cm sheep skin tissue areincubated in 10 ml 0.25 percent glutaraldehyde solution in PBS (pH 7.2)for 24 h. The fixed tissue pieces are washed with PBS several times,wiped clean and stored in PBS in refrigerator until use. Theglutaraldehyde treated tissue as above is used as a model substrate forcreating porosity and forming implant in the artificial porosity. Theglutaraldehyde treatment stabilizes the tissue and enables the tissue tobe used in drug delivery experiments without contamination anddegradation. 100 mg of Poly(dl-lactide-co-polyglycolide) (PLGA) (50:50),0.2 inherent viscosity 0.2 dl/g from Purac Biomaterials, Lincolnshire,USA (catalog name PURASORB PDLG 5002) is dissolved in 900 mg dimethylsulfoxide (DMSO). 0.8 ml of this PDLG 5002 solution is then mixed with8.0 mg moxifloxacin base as an exemplary drug and 5 microliter ofmethylene blue solution (10 mg in 2 ml DMSO, is added as an exemplarycolorant). The solution is transferred to 3 ml glass syringe. A 3 by 3hollow microarray is (33 MP) is attached to the syringe and the bluesolution of PDLG in DMSO is injected in the tissue. The array needlesare inserted completely and then withdrawn/pulled out to a predeterminedlength (80 percent of needle length) and then syringe is pressed toinject solution. The injection process is done at 15 different locationson the 2 cm by 2 cm glutaraldehyde fixed tissue. The excess solution iswiped off from each injection to remove any surface contamination. Thewater soluble DMSO is dissipated in the tissue, precipitating the waterinsoluble PLGA polymer in the artificial cavities. Total 135 micro PLGAimplants with encapsulated moxifloxacin (9 times 15) are created insitu. The infused sample is placed in 3 mL of PBS (containing 0.02%Na-azide as preservative, pH 7.4). The PBS is collected at each timepoint and fresh PBS is added. The release is monitored several days. Thecollected PBS is analyzed using a UV spectrophotometer (wavelength 294nm) to determine the moxifloxacin base concentration that is eluted fromthe tissue sample at the various time points.

In another variation of this method, a 3 ml suspension (60 mg ofmicrospheres suspended in 3 ml glycerol) comprising PLGA microspheres(Rifampin encapsulated in PDLG 5002, 10 percent rifampin loading,average size 10-100 microns), is filled in the 5 ml syringe. The arrayneedles are then pressed in tissue completely forming cavities. Thesyringe is then pushed to force the suspension via hollow needles intothe tissue cavities. The array is pulled back and excess suspension iswiped off with the tissue paper. The glycerol is dissipated in thetissue leaving behind the microspheres inside the cavities. The rifampininside the microparticles is released and is monitored over several timepoints.

Example 14B

Array formed using two step method: creating artificial cavities in thetissue and then infusing the injectable drug delivery composition in thecavities

The porosity in the skin tissue is first created by pressing 3 by 3hollow microneedle (33 MP) array at 15 different locations on the 2 cmby 2 cm tissue as above (135 total cavities). The PLGA and moxifloxacin,methylene blue solution in DMSO as above is poured on the treated porousarea and incubated for 5-30 minutes. The excess surface solution iswiped off with tissue paper. After dissipation of DMSO by tissue, theinfused solution is converted into precipitated polymer in theartificial pores created by the array microneedles. The release ofprofile of moxifloxacin from the formed PLGA microimplants in the cavityis monitored over several time points.

Example 14C

In situ drug delivery array formed by inserting the porosity creationneedle/array via injectable composition or polymer solution layer ontissue.

The PLGA, methylene blue and moxifloxacin solution in DMSO as above ispoured on the tissue first to form a liquid solution layer on the tissuesurface. A 3 by 3 hollow microneedle array (33 MP) is pressed on thetissue via polymer solution layer. As the array needles penetrate thetissue and they form cavities inside the tissue, the solution that isadhered to the needles surface is carried away in the cavities duringinsertion process (9 injections, 135 total cavities). After completeinsertion, the array is removed from the surface and excess surfacesolution is wiped off with a tissue paper. The infused solution in thecavity is converted into precipitated polymer in the artificial porescreated by the microneedles. Care is taken that no surface polymer ispresent on the tissue surface. The release of profile of moxifloxacinfrom the formed PLGA implants in the cavity is monitored over severaldays.

In another variation of this method, a suspension comprising PLGAmicrospheres (Rifampin encapsulated in PDLG 5002, 10 percent rifampinloading, average size 10-100 microns, suspended in glycerol) is appliedinstead of PLGA polymer solution. The rifampin encapsulated microspheresare made per Example 3 or are obtained by spray drying method. The arrayis then pressed in tissue forming cavities and pushing the microspheresinside the cavity. The array is pulled back and excess suspension iswiped off with the tissue paper. The glycerol is dissipated in thetissue leaving behind the microspheres inside the cavity. The rifampininside the microparticles is released and is monitored over several timepoints.

Example 15

Cell Encapsulated Microimplant Array Formed In Situ in the Tissue

Example 15A

Array formed by direct injection of injectable composition via hollowmicroneedle array. All the experiments in involving live cells andanimal experiments are generally carried out in sterile environmentunless mentioned otherwise (use of sterile hood and space; use ofsterilized plastic and glass ware; sterile filtered liquid, tissueculture mediums and solutions; sterile handling techniques and thelike). Ten pieces of 2 cm by 2 cm freshly procured sheep skin tissue areused in this experiment. The tissue is sterilized by incubating in 70percent ethanol for 30 minutes followed by three 10 minutes incubationsin sterile PBS. Chinese hamster ovary (CHO) cells (a cell line derivedfrom the ovary of the Chinese hamster) is prepared for encapsulation inthe array. Chinese hamster ovary (CHO) cells (supplied by ATCC, CHO-K1(ATCC® CRL-9618) are thawed to 37 degree C. and transferred to a 75centimeter square tissue culture flask containing 20 ml of ATCCformulated F-12K Medium with fetal bovine serum (final concentration of10%). The cells are incubated at 37° C. in a suitable incubator thatprovides 5% CO2 in air atmosphere. The medium is changed daily. After2-3 days and reaching full confluence, the cell culture medium isremoved and cells are rinsed with 0.25% trypsin, 0.03% EDTA solution.Additional 1 to 2 mL of trypsin-EDTA solution is added and is incubatedat 37 degree C. until the cells detach. The cells are centrifuged,supernatant is removed. Fibrin glue components from EVICEL® or TISSEEL(2 ml final volume) are mixed as per manufacturer instruction andtransferred to sterile 5 ml centrifuge tube. The fibrin glue componentsare then mixed with CHO-K1 cell suspension (30 microliters, 100000cells, exemplary live cell suspension). The mixture is vortexed andquickly transferred to 3 ml syringe. A steam sterilized 3 by 3 hollowmicroarray is (33 MP) is attached to the sterile syringe containingcells and fibrin glue component. The microarray needles are insertedinto the tissue completely, the needles are pulled back about 90 percentfrom the tissue and the created cavity is injected with fibrin glue andcell suspension. Care is taken that the fibrin glue is not prematurelycrosslinked or gelled before injecting it into the tissue (fibrin gluegel time 1-2 minutes depending on formulation used). Those skilled inthis art understand that Chinese hamster ovary (CHO) cells used in thisexample is for illustration only and other mammalian cells can also beused in place of CHO cells. The fibrin glue crosslinks along with thecells in the cavities in the tissue. Upon crosslinking, the array iswithdrawn from the tissue leaving behind gelled fibrin glue sealantmicroimplant array with live cells. The cells used here are forillustration only to show that cells can be entrapped without losingviability. The cell viability in the deposited materials can be checkedby cutting the treated area and using cell live-dead assay for cellviability. The viability of cells is checked in the fibrin gel usinglive-dead cell assay (Acridine orange and propidium iodide as dyes). Thelive cells appear green and dead cells appear red when entrapped cellsare viewed under the microscope and using blue light for illumination.The cell viability also can be checked using tripan blue assay. The deadcells are stained blue and live cells appear transparent. Cell viabilityis generally found in the range of 60-90 percent in fibrin glue.

In another modification of above example, flask of fully-grown humanforeskin fibroblasts cells (ATCC sourced) are passaged in MinimumEssential Medium (MEM)/10% fetal bovine serum (FBS) are resuspended inMEM/10% FBS and approximately 100000 cells suspended in 0.2 ml sterilePBS or MEM are carefully transferred in the 5 ml sterile tube containing2 ml fibrin glue components (fibrinogen and thrombin the majorcomponents mixed together). The fibrin glue precursors and HFF cellsuspension is injected via 33 MP array in the tissue as above and uponcrosslinking 2-10 minutes, the array is removed leaving behind thecrosslinked fibrin glue in the cavities created by the array. The sameexperiment can be done on live mouse tissue. The back surface of themouse is shaved, sterilized with iodine and then fibrin glue and mousefibroblasts are then injected in the skin tissue as above. For humans,fibroblast cells from suitable donor or autologous HFF cells may beused.

In another experiment as above, fibrin glue is substituted withsynthetic biodegradable crosslinker precursors based on PEG-lactateacrylate macromonomer is used. 2 g of PEG-LACTATE-5-acrylate preparedper Example 10D is dissolved in 8 g PBS and sterile filtered. To thissolution, sterile filtered eosin y (final concentration 20 ppm), vinylpyrrolidinone (10 microliter/ml final concentration) and triethanolamine (final concentration 90 mM) are added. 100000 HFF cells suspendedin 0.2 ml sterile PBS or MEM are carefully transferred to themacromonomer solution and vortexed. The cell suspension is infused using33 MP array as above in dermal tissue or live tissue. The array needlesare removed and the treated are skin is exposed to 512 nm laser(intensity 10-100 mW per centimeter square) light or high intensitywhite floodlight. The cell suspension undergoes polymerization andcrosslinking without damaging the cells. In another embodiment, 36microneedle array is used to make 36 cavities first and then fibrin gluecomponents and cell mixture is infused in the cavities. The excesssolution is wiped off and the solution in the cavities undergocrosslinking and form a fibrin clot inside the cavities forming amicroimplant array with cells with fibrin sealant as encapsulationmatrix.

In another variation, thermoreversible gelatin solution is used as acell carrier. 1 g gelatin is dissolved in 9 ml PBS and the solution iswarmed to 60-70 degree to dissolve the gelatin. The solution is cooledto 37 degree C. 2 ml of this solution is mixed with 0.2 ml sterile PBScontaining 100000 HFF cells suspended. The warm mixture is loaded insyringe, attached to 33 MP injector. The microarray needles are pushedin the sheep skin tissue and injected with gelatin solution with cells.

The injector is left in the skin for 30 minutes. A small portion of iceis applied to accelerate gelation of gelatin. After gelation of gelatinin the cavities, the array is withdrawn leaving behind the gelatin gelwith encapsulated HFF. Other thermosensitive gel polymer such asPluronic or PEG based polymer or n-isopropylacrylamide based polymers orcopolymers may also be used in place of gelatin as long as they do nothave components that are toxic to cells.

Example 15B

Array formed using two step method: creating artificial cavities in thetissue and then infusing the cell encapsulation compositions.

The porosity in the skin tissue as used in is first created by pressing3 by 3 hollow microneedle (33 MP) array at 15 different locations on the2 cm by 2 cm tissue as above (135 total cavities). The fibrin glue withcells suspension or PEG-LACTATE-5-acrylate solution with cells orgelatin solution with cells is applied on the tissue where cavities arecreated. The solution is incubated for 2-5 minutes. Air jet may be usedto aid in filling the solution in the cavity. Excess solution is wipedoff and the solution in the cavity allowed to gel (exposed to visiblelight for PEG-LACTATE-5-acrylate solution). The gelled solution in thecavity entrap cells for local or systemic therapeutic effect.

Example 15C

In situ drug delivery array formed by inserting the porosity creationneedle/array via cell encapsulation compositions comprising cells.

A gelatin or fibrin glue or PEG-LACTATE-5-acrylate solution with cellsis first applied on the tissue to form a layer and then 3 by 3 hollowmicroneedle array (33 MP) is pressed on the tissue through the cellsuspension. As the array needles penetrate the tissue and they formcavities inside the tissue, the solution that is adhered to the needlessurface is carried away in the cavities during insertion process (135total cavities). After complete insertion, the array is removed from thesurface and excess surface solution is wiped off with a tissue paper.The infused solution is crosslinked inside the cavity and entrapping thecells.

Example 15D

Delivery of Cells Using Oscillating Needle

From a rat biopsy sample, rat cells (fibroblasts) are isolated, expandedin a culture dish and resuspended in a 1 ml PBS buffer (about 1 millioncells in one ml PBS). The cell suspension is infused in the same ratskin using an oscillating needle as described before. The oscillatingneedle helps to infuse the cells in large surface are uniformly asopposed to single injection of cell suspension using a syringe andneedle. This uniform distribution of cells using oscillating needledevice can be beneficial in many stem cells based therapies.

Example 15E

Microimplant arrays containing live cells made using blood plasma basedbiodegradable hydrogels. Porcine pericardial tissue were cut in 1 inch×1inch dimension and vacuum dried for 2 days. The dried tissue is cut in 1cm×1 cm pieces and 9 holes (3×3 arrays) are made using 21 G needle. Thetissues with cavity are then placed on a thin glass slide. The 3T3 cellsare cultured for 5 days in a standard cell culture medium and cellculture flask. The cells are trypsinized and centrifuged. Thecentrifuged cells are resuspended in 0.05 ml sterile porcine plasma andmixed with 10 microliters 20% triethanolamine solution. To thissolution, 50 microliters of 30% PTE-050GS glutarate NHS estercrosslinker solution in PBS (20 mM pH 7.2). Crosslinker PTE-050GS ispurchased from NOF Corporation, Tokyo, Japan; molecular weight 5000g/mole, 4 arm star shaped, with terminal NHS groups and glutarate asdegradable ester. The mixture is added to 9 cavities of array, excesssolution is wiped from the tissue surface and the mixture is allowed toform a crosslinked gel. The crosslinker reacts with the plasma proteinsand form a crosslinked gel in 10-30 seconds. The crosslinked gel withlive cells are stained using Tripan blue stain solution. The live anddead cells are counted using microscope in the array at 10 randomlocations. The array gels had 25±10 live cells and 1±1 dead cells. Thehigh number of live cells indicate tolerance of crosslinking reactionconditions by the cells entrapped in the gel array. It also indicatesthe successful formation of live cell arrays in the tissue

Example 16

Preparation of Microneedle Array Comprising Cells.

Chinese hamster ovary (CHO) cells (supplied by ATCC, CHO-K1 (ATCC®CRL-9618) are thawed to 37 degree C. and transferred to a 75 centimetersquare tissue culture flask. containing 20 ml of ATCC formulated F-12KMedium with fetal bovine serum (final concentration of 10%). CHO cellsare used as an illustrative mammalian cell line. The cells are incubatedat 37° C. in a suitable incubator that provides 5% CO2 in airatmosphere. The medium is changed daily. After 2-3 days and reachingfull confluence, the cell culture medium is removed and cells are rinsedwith 0.25% trypsin, 0.03% EDTA solution. Additional 1 to 2 mL oftrypsin-EDTA solution is added and is incubated at 37 degree C. untilthe cells detach. The cells are centrifuged, supernatant is removed andcells are resuspended 5 ml PBS containing 10 percent DMSO ascryopreservative agent. Silicone base MPatch™ Microneedle templates areprocured from Micropoint Technologies Pte Ltd. (Singapore). The mold hasfollowing characteristics: 20 mm dia and 4 mm height. 10 by 10microneedle 700 microns height pyramidal shaped cavities with 200microns by 200 microns base and 500 microns pitch. The mold is washedwith mild soap, sterilized using 70 percent isopropanol and used toprepare cell containing microarray. The PBS containing cell suspensionis added on top to mold cavities until all mold cavities are filled withthe PBS. The mold is centrifuged at 1000 rpm to drive occupy cellsuspension all the space in mold cavity. The mold is rapidly cooled to−80 degree C. to freeze the PBS and cell suspension in mold cavity. Uponcomplete solidification of PBS solution, the array is stored at −192 orat liquid nitrogen temperature to preserve cell viability. Before use, asticky tape is applied on the base of the needle with needles stickingout of surface. The array is removed immediately used to insert in thesheep skin tissue at room temperature or live skin tissue. The needlesundergo melting at room temperature inside the tissue releasing thecells in cells which get nutrients and other essential environmentinside the live tissue environment. The viability of cells inside thetissue is checked using live dead assay as mentioned before.

In another modification of above example, ATCC formulated F-12K Mediumwith fetal bovine serum is used in place of PBS.

In another modification of above example, human foreskin fibroblasts(HFF) cells in PBS are used in place of CHO cells.

In another modification of above example, human foreskin fibroblasts(HFF) cells in fibrin glue precursors (before crosslinking) are used inplace of PBS. This produces frozen array with fibrin glue precursors.The cells stay entrapped in fibrin glue which get crosslinked uponinsertion in the body under physiological conditions (pH 7.4, 37degree).

In another modification of above example, PEG based precursors (Example10D, 10E, 15), DMSO as a cryopreservative and cells are encapsulated ina hydrogel. The precursors are cast and crosslinked in array format withlive cells. The array is stored with live cells at −192 degree C. inliquid nitrogen until use. Prior to use, the array is inserted in thetissue at around −20 degree C. in frozen state where the hydrogelprovides mechanical integrity to the cells and DMSO provides protectionfrom freezing during storage.

Example 17

Drug Release from Implanted Arrays

Example 17A

Bupivacaine controlled release from the in situ created microimplantarray prepared using oscillating needle and PLGA polymer solution.

In another embodiment, bupivacaine releasing microimplants array isprepared using oscillating needle to create porosity and infuse polymersolution in the porosity. Briefly 172 mg PLGA polymer (PDLG 5002) isdissolved in 1.75 ml DMSO. 0.75 ml of polymer solution and 23 mg ofbupivacaine base is mixed, the solution is applied on the glutaraldehydefixed bovine pericardium tissue. A commercial tattoo machine needle(permanent make up machine needle) is used to create porosity and drivethe solution inside the tissue. The needle is moved on one squarecentimeter area. The machine needle oscillated at 6000 minutes perminute. After about one minute, the machine is stopped and excessbupivacaine solution is wiped off from the tissue surface. Care is takento ensure that no polymer sample is precipitated on the tissue surface.A control sample is prepared/tattooed using identical conditions whereonly polymer solution in DMSO without drug is used for infusion. Thetreated areas (bupivacaine treated and polymer treated control) are cutfrom the tissue and are subjected to drug release in PBS at 37 degree C.for several days. The concentration of bupivacaine in the eluted samplesis monitored using UV spectrophotometer (absorbance monitored at 262.5nm). A bupivacaine base release profile elution curve is shown in FIG.13 along with polymer. The microimplants created by oscillating needlesuccessfully penetrated the tissue and infused the drug in the tissue.The DMSO is dissipated in the tissue leaving behind PLGA polymer alongwith hydrophobic bupivacaine base. The release from the precipitatedpolymer is shown in FIG. 13. It is clear from the FIG. 13 that thebupivacaine is released in a sustained manner for few days.

Example 17B

In situ created micro array implant containing drug encapsulatedmicrospheres.

Microarray implants filled with rifampin encapsulated microspheres.

Rifampin containing red/yellow microspheres are prepared using spraydrying method or prepared according to Example 3. Microspheres areprepared using PLGA polymer (PDLG 5002 from Purac) polymer with 20percent rifampin loading relative to the polymer plus drug weight.Control PLGA microspheres without rifampin are prepared under identicalspry drying conditions. 4.9 mg of rifampin encapsulated microspheres aresuspended in 0.25 ml glycerol. One/two drops of the suspension are addedon the 2 by 2 cm glutaraldehyde fixed sheep tissue. The 33 MP microarrayneedles are pressed on the tissue via rifampin suspension to create 3 by3 micro array cavities and infuse the cavities with the rifampinmicrospheres. The procedure is repeated 9 times on the same tissue atdifferent locations to create 135 micro cavities loaded with rifampinmicrospheres. The excess suspension is wiped of using tissue paper. Noloosely adherent microspheres in the tissue are noticed. The glycerol inthe suspension is dissipated in the tissue leaving behind themicrospheres in the cavities. Identical procedure is used to incorporatecontrol microspheres without drug. The rifampin treated tissue andcontrol tissue are incubated at 37 degree C. in 3 ml PBS and rifampin intreated and control samples is monitored at various time points.Rifampin concentration in the eluted samples is analyzed using UV-VISspectrophotometer (wavelength 474 nm). Release of rifampin from thetreated tissue and control sample is shown in FIG. 14. The treatedsamples show a sustained release of rifampin from the in situ formedmicro array implants and as expected, control samples did not show anysignificant release of rifampin.

Example 18

Microencapsulated Islets in Sodium Alginate

Immunoisolation of Islets in Alginate Crosslinked Hydrogels

1.6 g sodium alginate (molecular weight 277000 g/mole, 67 percentguluronic acid) content is dissolved 100 ml HEPES buffer pH 7.4.Separately freshly obtained islets are centrifuged and the pellet iswashed with HEPES buffer. The process is repeated until pellet is freefrom calcium ion from the culture media. 1 ml of the alginate solutionis mixed with rat islet cell suspension (10000 islets) and mixedthoroughly. The cell suspension is fed via a syringe pump and atomizedthrough an atomizing apparatus. The condition of the apparatus (airpressure, nozzle diameter and the like are adjusted to get 100-500micron size droplets) and are collected in 1.1 percent calcium chloridesolution to form calcium alginate microspheres containing live isletcells. The microspheres are filtered and washed with 0.5 and 0.25percent calcium solution and incubated for 3 minutes in CHES-buffered1.1 percent calcium chloride, and again washed with 1.1% calciumchloride solution. The microspheres are incubated in poly (L-lysine) (a0.1% solution of molecular weight 20100 g/mole; in10 mM HEPES-bufferedsaline, pH 7.4, Sigma) for 8 min and subsequently washed withHEPES-buffered saline (HBSS). The viability of the islets is checkedusing tripan blue assay. For additional information onmicroencapsulation of islets, please refer to E. C. Opara et al.;Methods Mol Biol., Volume 1001, page 261-266 (2013), cited herein forreference only.

The islets containing microspheres are inserted in 1000 micron dia and600 micron height cavities (36 cavities) in the sheep skin tissue andthe cavity is sealed using fibrin sealant or DuraSeal sealant. Thediameter of encapsulated cells microparticles must be less than thecavity size diameter to ensure entry of cells in the cavities.

Example 19

Formation of implanted array in the tissue using “array in array”device.

Example 19A

External formation cylindrical microimplant suitable for implantingusing “array in array” device and implanting them to form a drugdelivery array.

25.2 mg bupivacaine base, 49.9 mg PDLG 5004 polymer and 0.45 mltetrahydrofuran (THF) are mixed to prepare a bupivacaine base coatingsolution (copolymer of DL-lactide and Glycolide in a 50/50 molar ratioand with an inherent viscosity midpoint of 0.4 dl/g). 10 mg bupivacainebase, 49.8 mg PDLG 5004 polymer (PLGA copolymer 50:50 lactide toglycolide ratio) and 0.45 ml tetrahydrofuran are mixed to prepare abupivacaine base coating solution (20 percent drug solution). 49.9 mgPDLG 5004 polymer and 0.45 ml tetrahydrofuran are mixed to preparecontrol coating solution with no drug (control solution). Six 30 cmlong, 100 micron diameter twisted submucosa tissue threads are washed 3times using distilled water and then finally with acetone and dried atroom temperature for 24 h. 2 pieces each are incubated for 5 minutes in50 percent, 20 percent and control solutions, dried at room temperaturefor 15 minutes. The process is repeated three times and coated threadsare dried under vacuum for 30 minutes. One piece of thread is incubatedin 3 ml PBS (pH 7.4) and bupivacaine base release is monitored at 37degree C. for 5 days. Fresh PBS is exchanged for at every time point andthe bupivacaine concentration is monitored using UV spectrophotometer(absorbance measurement at 262.5). Release profile for other threads isobtained using same procedure. Bupivacaine base release from the control(triangles), 20 percent bupivacaine coating (solid circles) and 50percent bupivacaine coating (rectangles) is shown in FIG. 17. Asexpected, the control sample did not show any release of bupivacaine.The threads coated with 20 and 50 percent solution provides sustainedrelease of bupivacaine upto 72 hours. The coated threads as above arecut using microtome machine to produce coated microcylinders(approximate 100 mm diameter and 1000 micron length). The cutmicrospheres are placed in hollow cavities of microneedle array (similarto “base array” as described in FIG. 6A, 10×10 hollow stainless steelmicroneedle array with 1200 mm needle ID, 1200 mm needle length, 1 mmdistance between each needle). The array needles are inserted in theporcine skin tissue. Using a wire/rod (1000 micron OD), eachmicrocylinders is pushed out of the hollow cavity in to the tissue.After pushing all the 100 microcylinders, the array is pulled out fromthe tissue leaving behind 100 microimplants in an array format in thetissue. A total of 300 microimplants are inserted which provides acumulative length of 30 cm thread which is used in drug release study.The porcine tissue is cut and bupivacaine release is monitored as above.

Example 19B

Casting of implants in “array in array” device and implanting them inthe skin tissue In another modification of above embodiment, the needlesof the 10 by 10 array as used in Example 19A are inserted in 0.5 mmthick leather sheet. The drug/polymer coating solution as above ispoured into the hollow cavities of the array. Since the other end ofneedles are blocked using a leather, the solutions pools into thecavity. Excess solution is wiped off and the solvent is evaporated undervacuum leaving behind PLGA implants with bupivacaine in the cavity. Thearray is removed from the leather and is then inserted in the porcinedermal tissue. The cast PLGA implants in the hollow cavity of the arrayare then pushed out in the skin tissue using a wire/rod as above to formPLGA based biodegradable polymer implant array. In another modificationof above example, THF in the coating solution is replaced with dioxaneand the solution is poured in cavities of the array and cooled to −5degree C. and lyophilized to remove dioxane completely, leaving behindthe lyophilized PLGA implant in the array cavity. The lyophilizedimplant is inserted in the skin tissue to form a microimplant array asdiscussed previously.

Example 20

Biodegradable Arrays Made from Crosslinked Synthetic BiodegradablePolymers.

Example 20A

Biodegradable Microneedle Made from Polyethylene Glycol BasedMacromonomers

Use of UV Initiated Polymerization of Biodegradable Macromonomers toProduce Biodegradable Implantable Microarray with or without Drug

3 g of PEG-LACTATE-5-acrylate diacrylate prepared per Example 10D isdissolved in 9 g PBS. 300 mg Irgacure 2959 is dissolved in 700 mgn-methyl pyrrolidone. 50 microliter of Irgacure 2959 solution is addedto the PEG-LACTATE-5-acrylate solution and 100 mg heparin as model watersoluble drug is added to the solution. The solution is transferred to a50 ml Schlenk tube and subjected to three freeze-thaw cycles undervacuum 3 times to remove dissolved gases from the solution. The solutionis sterile filtered using 0.2 micron filter. The sterile solution(precursor solution) is filled inside sterile cavities of MPatch™Microneedle array. The excess solution is wiped off from the moldsurface and spun for 5 minutes at around 5400 rpm. The solution isexposed to long UV ultraviolet light (Black-Ray UV lamp, 360 nm light,10000 mW/cm2 intensity) for 5 minutes to photopolymerize and crosslinkthe infused precursor in the array cavities. The crosslinked array isair dried. A polyester one side adhesive tape is attached to the base ofdry crosslinked PEG gel and the array is removed from the mold cavity.The array has sharp needles which can be inserted in the skin or tissue.In one embodiment, array is made with no drug.

To further improve mechanical strength and hardness of tip, theprecursor array solution is mixed with biocompatible and biodegradableinorganic or organic filler materials, especially at the tip of thearray are used. In one embodiment calcium carbonate or magnesiumcarbonate powder (size less than 300 microns) is used. Two precursorsolutions are prepared. One solution has 100 percent (relative toprecursor macromonomer weight) magnesium carbonate is added and theother solution did not have any magnesium carbonate added. The magnesiumcarbonate precursor solution is added into mold, crosslinked via UVinitiated polymerization, dried and the array is produced. This arrayhas 20 percent magnesium carbonate fine particulate salt added asbiocompatible biodegradable filler to increase stiffness of the array.In separate experiment, the precursor with magnesium carbonate is addedfirst and added only upto 10 percent of the cavity volume is filled (tipof the array) and spun for 2 minuets. The second solution withoutmagnesium carbonate is then added to the array cavity on top of thefirst solution and cavities are filled completely. The excess solutionis wiped off and the mold is spun for 5 minutes and then exposed tolight. The precursor undergoes polymerization. The crosslinked productis dried in the mold and removed with the polyester tape backing asabove. The crosslinked array has magnesium carbonate reinforced tip atthe bottom of the tip which helps to insert in the skin tissue.

In another embodiment, magnesium carbonate in the above is replaced withrifampin loaded microspheres. The biodegradablemicroparticles/microspheres serve as a reinforcement of the crosslinkedhydrogel matrix as well as sustained release carrier for drugs. Inanother embodiment, sodium hyaluronate or other water solublebiocompatible polymers are added to reinforce the gel for improvedmechanical properties.

Example 20B

Use of Visible Light Initiated Polymerization of BiodegradableMacromonomers to Produce Biodegradable Implantable Microarray with orwithout Drug

In 100 ml beaker 3 g of PEG-LACTATE-5-acrylate diacrylate prepared asabove is dissolved in 9 g PBS. In another 10 ml glass vial, 300 mg eosinY is dissolved in 700 mg n-vinyl pyrrolidinone. 30 microliter of eosin ysolution, 1 ml of 5 M triethanol amine in PBS are added toPEG-LACTATE-5-acrylate solution. The solution is degassed using threefreeze thaw cycles. (the solution is cooled in liquid nitrogen andvacuum is applied and the frozen solution is warmed at room temperatureunder vacuum). The solution is sterile filtered and protected from lightusing aluminum foil. The precursor solution is added on top of MPatch™Microneedle array mold and spun for 10 minutes (5400 rpm) to infuse thesolution completely in the mold cavities. The infused solution iscrosslinked by photopolymerization by exposing it to 512 nm laser (argonlaser, intensity 100 mW per centimeter square) light or high intensitywhite floodlight. The light polymerizes and crosslinks thePEG-LACTATE-5-acrylate monomer and forms a gel in the mold. The gel inthe mold is air dried. A polyester adhesive tape is attached to the baseof the array needles and the array is removed gently from the mold. Thearray has sharp tip and is biodegradable if implanted in the skintissue. Cells or drugs may be added to crosslinked gels for sustainedrelease. Also, biodegradable microparticles/microspheres with or withoutdrug encapsulation may be added (5 to 80 percent of gel volume) in thearray.

Example 20C

Synthetic Biodegradable Crosslinked Hydrogel Based Arrays forImplantation.

Use of PEG Based Crosslinked Hydrogels.

PEG based crosslinked hydrogel arrays made by condensationpolymerization reaction of precursors comprising nucleophilic andelectrophilic reactive groups.

In a 50 ml beaker, 1 g of tetrafunctional star shaped amine terminatedpolymer, molecular weight 10000 g/mole with terminal amine groups(available from Laysan Bio, Inc. AL, PEG10K-4Amine) is dissolved in 4 gPBS (20 mM, pH 7.4). Separately, 1 g of PEG10KARM glutarate NHS ester isdissolved in 4 g PBS (20 mM pH 7.6). 20 microliter of above aminesolution is mixed with 20 microliter solution of NHS ester and the geltime is checked. If the gel time is too fast (within 30 seconds, pH ofthe PBS used in PEG NHS ester is changed to mildly acidic to mildlybasic region (pH 6.8 to 7.8). If too slow polymerization orcrosslinking, the pH is adjusted to basic side (upto pH 7.8). The pH canbe adjusted using 1 N HCl or 1 N NaOH. The gel time is adjusted to about60-120 seconds. Both solutions are sterile filtered, degassed and thenmixed and filled immediately in the mold and spun for 2 h and then airdried. The crosslinked gels array is attached to polyester tape withadhesive and is removed from the mold. The array has PEG based syntheticcrosslinked hydrogel which can be inserted in the tissue.

The array can be reinforced at the tip using magnesium carbonate as afiller as described above. The PDLG 5002 based biodegradablemicroparticles/microspheres may be added with or without drug as amechanical reinforcement agent and/or sustained drug release agent. Thebiodegradable microparticles may be added at 5 to 80 percent of gelvolume in the array. In another embodiment, sodium hyaluronate or otherwater soluble biocompatible polymers are added to reinforce the gel forimproved mechanical properties.

Example 20D

Array produced using melted polyethylene glycol polymer with or withoutreinforcing agent Polyethylene glycol, molecular weight 35000 (PEG35K)is used in this experiment. 5 g of PEG35K and 20 mg rifampin are mixed.The polymer is melted until drug is completely dissolved, degassed themelted polymer using three freeze-thaw cycles and poured into siliconrubber mold as used above. The mold is preheated to 60 degree to avoidpremature cooling inside the cavity. The melted polymer is added in themold cavities and excess polymer is wiped off. The mold is then spun at5400 rpm 60 degree for 2 hours under heated chamber maintained at 60degree for 1 hour and cooled. The base material is attached with thepolyester tape and the PEG based array is removed from the mold. Themelted material is also degassed under vacuum prior to use. The meltedpolymer has higher density and sharp tips. The array can be inserted inthe skin where PEG is dissolved in the tissue producing a cavity.

In another variation of above embodiment, rifampin encapsulated PDLGbased microspheres are added 20 percent of the weight of PEG (weightpercent can be varied from 1 to 80 percent of the PEG, preferably 5 to50 percent). The rest of the procedure is same as before. This producesmelted PEG based microarray with drug encapsulated microspheres. Inanother variation, PEG is added with sodium chloride powder (pre sievedand used 300 microns or less fraction, weight percent may vary from 5 to80 percent) as reinforcing agent for PEG polymer. Other water solublebiocompatible inorganic or organic solids (sugars, amino acids and thelike) may also be used as filler. The use of inorganic filler increasesstiffness and improves use of insertion. In another variation, aninjection molding method is used to prepared PEG based arrays. Inanother variation, the same material is used as a base material insteadof tape. The inserted material in contact with the tissue is dissolvedleaving behind the base material.

In another variation of this embodiment, Pluronic F127, Pluronic F68,Tetronic 908, Pluronic F108 is used as water soluble low meltingpolymer.

In another embodiment, an exemplary polyethylene glycol-co-polylactonecopolymer, PEG-polylactate polymer that is water soluble and low melting(melting point less than 70 degree C.) is used to prepare the array.

Example 21

Implanted arrays made islets of langerhans.

Example 21A

Preparation of Array Containing Islets of Langerhans in the Skin TissueUsing Two Step Methods

Preparation of Artificial Porosity Using Dissolvable Array or RemovableArray

Sodium hyaluronate based dissolvable array is a gift from MicropointTechnology. The array is applied using an applicator provided by themanufacturer. On a shaved sheep skin, the 20 microneedle array (FIG.22A) is applied on the tissue to create multiple cavities. The needlesof the array penetrate the skin and produce micro cavities replicatingthe shape of array needles. Rat islets are isolated from rat pancreastissue are cultured in RPMI 1640 medium (GIBCO) (10 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES buffer with10% fetal bovine serum and 1 percent antibiotic-antimycotic mixture).Separately, in a 100 ml beaker 3 g of PEG-LACTATE-5-acrylate diacrylateprepared per Example 10D is dissolved in 9 g PBS. In another vial, 10 mlglass vial, 300 mg eosin Y is dissolved in 700 mg n-vinyl pyrrolidinone.30 microliter of eosin y solution, 1 ml of 5M triethanol amine in PBSare added to PEG-LACTATE-5-acrylate solution. The solution is sterilefiltered and protected from light using aluminum foil. About 1000 isletcells maintained in culture media are centrifuged at 100 g for 1 minuteto produce a pellet. The supernatant media is removed and the islets areresuspended in one ml sterile PEG macromonomer solution containing eosinand triethanol amine. The fluid from the porosity created in the skintissue is aspirated and the islets suspended in macromonomer solution isapplied on the area where porosity is created. The solution is incubatedfor 2-10 minutes and islets are allowed to infuse in the artificialcavities. The cavity size (average diameter) is substantially higherthan the size of islet cells. The excess solution from the skin tissueis wiped off and the infused solution is crosslinked byphotopolymerization by exposing it to 512 nm laser (argon laser,intensity 100 mW per centimeter square) light or high intensity whitefloodlight. The light polymerizes and crosslinks thePEG-LACTATE-5-acrylate monomer and forms a gel in the mold entrappingthe islets of Langerhans in the gel. The polymerized gel is permeable totissue fluids and nutrients as well as insulin. The treated area isfurther treated with macromonomer solution without islets to seal thetreated area. Alternatively, fibrin sealant can be used to seal thetreated area. The treated section is cut and viability of langerhans ischecked using standard live-dead cell assay available from MolecularProbe or Thermo Fisher Scientific. The live cells appear green and deadcells appear red when viewed using microscope.

An identical experiment is conducted on live rat tissue. Briefly ratsare made diabetic by injecting 50 mg/kg strepto-zotocin dissolved in acitric acid buffer solution (pH 4.5). Rat with blood glucose levels 300mg/dL are used for the study. Diabetic rats are anesthetized and shavedon the back. The porosity is created on the shaved area as above and ratislets are infused in the porous cavities. Three groups treatment arecreated containing 5 animals each. One group received the porositytreatment using dissolvable microneedles but no islet infusion (controlgroup). The second group (control B) are treated with dissolvablemicroneedles followed by islet solution incubation for 5 minutes. Theexcess solution is wiped of and the treated area is sealed using PEGmacromonomer solution and polymerized as above. Control B has islets inthe artificial cavity but no encapsulation matrix to protect againstimmune cells. In the third group (treatment group), the islets areinfused in the cavity along with macromonomer solution as above andsealed using the same macromonomer solution. The treatment groups hadislets encapsulated in immunoprotective gel made of PEG macromonomers.Control B and treatment group received approximately 2500 islets perrat. Glucose levels are monitored each rat in and all groups aremonitored for one month. Other method of cavity creation methodsdescribed in this invention in place of dissolvable array to createcavities amy also be used.

Example 21B

Preparation of Array Containing Islets of Langerhans in the Skin TissueUsing Two Step Methods

Preparation of Artificial Porosity Using Hollow Needle Microarray andFilling the Cavity Using Islets of Langerhans.

In a 100 ml beaker 3 g of PEG-LACTATE-5-acrylate diacrylate prepared perExample 10D is dissolved in 9 g PBS. In another 10 ml glass vial, 300 mgeosin Y is dissolved in 700 mg n-vinyl pyrrolidinone. 30 microliter ofeosin y solution, 1 ml of 5 M triethanol amine in PBS are added toPEG-LACTATE-5-acrylate solution. The solution is sterile filtered andprotected from light using aluminum foil. About 5000 islet cellsmaintained in culture media are centrifuged at 100 g for 1 minute toproduce a pellet. The supernatant media is removed and the islets areresuspended in one ml sterile PEG macromonomer solution containing eosinand triethanol amine. The solution is loaded in sterile 3 by 3 hollowmicroneedle array (33 MP). On a porcine skin tissue, the array needlesare firmly pressed against the tissue and all needles are completelyinserted in the tissue. The needles are pulled back about 800 microns(about 80 percent of the needle height) and the polymer-islet suspensionis injected in the tissue to fill the cavity. This process is repeated15 times at different tissue locations creating 135 microneedle cavitiesfilled with macromonomer solution. The treated area is wiped off usingtissue paper to remove surface solution. The treated area is exposed toargon laser light (513 nm) for 2 minutes to crosslink the macromonomerin the solution. The treated area is covered with 0.2 ml macromonomersolution without islet cells and exposed with light to seal the treatedarea.

In another embodiment, alginate encapsulated islets are used in place ofislets alone. The alginate encapsulation matrix providesImmunoprotection to the islets.

Example 22

Microimplants Array Implanted Using “Array in Array” (AIA) Device

Example 22A

Fabrication of “Array in Array” Device.

Design 1

The 33 MP hollow microneedle device from Micropoint is used. The femalehub of the device is cut using a diamond cutting wheel at the base ofthe hub leaving behind 3 by 3 array needles attached to 1 mm thick disk.The needles are 1 mm in height attached to stainless steel 1 mm disk.This part serves as a base array as described in FIG. 6A. The hollowcavities of the needle are coated with silicone oil to help in castingprocess. 1 ml of EasyCast® Clear Casting Epoxy precursor resins aremixed (epoxy resin and amine hardener). The hollow metal part istransferred to a paper cylinder and the resin solution is poured overthe needles and its base. The resin is added to create a 1 mm thick diskupon casting. Upon curing, the cured resin disk and its array needlesare removed from the bottom hub. The solid needles of epoxy cast resinare polished to reduce its diameter so that it can easily be inserted inthe needles of hollow needle array. The cast portion of array representsplunger array as Described in FIG. 6B. In another variation, instead ofepoxy resin, a light curable dental cavity filling resin or ethyleneglycol dimethacrylate with Irgacure 2950 as UV light photoinitiator isused in place of epoxy resin. The monomer solution along with UVphotoinitiator is added on the mold as above and cured using 360 nm UVlight for 5 minutes. The cast array made of photocured methacrylatepolymer or dental resin can be used to push the implant out as describedbelow. In another example, two part silicone rubber precursor SYLGARD®184 (Dow Corning) is cast inside the array to make a rubber basedplunger array.

Design 2

AIA Array Made using Rigid Stainless Steel Base Plate

In another embodiment, an “array in array” device described according toFIG. 6 is made. The base array design is shown in FIG. 6A. A stainlesssteel plate, 1 mm thick and 20 mm length and 20 mm width is used. At thecenter of the plate 25 holes with 0.55 mm dia are drilled in the 5 by 5matrix format. 25 hollow needles 0.55 mm OD and 0.31 ID with sharpcutting edge are inserted in each hole. The needle length protrudedperpendicular to plate surface and 1 mm height (needle height) from thesurface. The needles are adhesively joined to the plate surface. 4 guideposts (2.5 mm dia cylinders) are also attached along the periphery andat the center of each side of the plate. The excess length from otherend of plate surface is cut and polished exposing the cavity holes onplate surface. FIGS. 15A and 15B show illustrative images of thefabricated base array with 25 hollow microneedles with 1 mm length and0.31 mm diameter.

The plunger array is prepared in a similar manner (Design shown in FIG.6B) as base array. A stainless steel rectangular plate, 3 mm thick and20 mm length and 20 mm width is used. At the center of the plate 25holes with 0.3 mm dia are drilled in the 5 by 5 matrix format. Thematrix position and format is same as base array needle format. 25hollow SS wires (OD 0.3) are inserted in the holes. The solid needlelength protruded perpendicular to plate surface and 1 mm height (needleheight) from the surface (total wire length including plate thickness is2 mm). The end of the wire is flat and does not have sharp edge. Thepurpose array is to push the implant from base array cavity volume. Theplunger needles are adhesively joined to the plate surface. 4 guideholes (2.6 mm dia) are also drilled along the periphery and at thecenter of each side of the plate (position identical to guide posts).The excess length from other end of plate surface is cut and polished.FIGS. 15C and 15D show the illustrative fabricated plunger array with 25hollow microneedles with 1 mm length and 0.3 mm diameter.

When plunger array is kept on top of base array without insertion, theposition of each array needle matches with each other and the plungerarray needles can insert inside the base array cavity needles (FIG.15E). A spacer plastic sheet, 2 mm thick, is kept on base array topsurface. The presence of spacer sheet prevents insertion of plungerarray needles into base array needle cavities. After removal of spacersheet, the aligned needles of plunger array enter the cavity ofcorresponding base array microneedle (FIG. 15F).

Design 3

Flexible AIA Array

In another variation of AIA apparatus, the stainless steel base plate inthe bottom and/or top array is replaced with flexible rubber/plasticlike material. A 1 mm thick polyurethane foam or polystyrene foam is cutinto 20 mm by 20 mm rectangular pieces. Twenty five 24 gauge syringeneedles are inserted in the foam in a 5 by 5 format with needle toneedle distance is kept at 2 mm. SYLGARD® 184 (Dow Corning), a twocomponent clear curable silicone elastomer is used. The base material ofSYLGARD® 184 is mixed with a curing agent in 10:1 (SYLGARD®-to-curingagent ratio). The mixture is then degassed for 10 min and added on topof foam and needle until (final liquid thickness 1 mm). The siliconerubber is cured for 48 h until soft elastic transparent rubber isobtained. The cured rubber along with its needle is lifted from thefoam. One side of the silicone rubber had 1 mm long 24 gauge needle in 5by 5 array format and other side has excess needle material which is cutalong the surface of the cut, thus creating 25 openings of cavities onrubber surface. Similarly, plunger array needles in the same pattern ashollow needle are cast using 0.3 mm stainless steel wire/rod with 1 mmneedle length and excess rod is cut from the rubber surface. The plungerarray needles fit inside the hollow cavity of base array. Siliconerubber being flexible, it can potentially help to use on curved surfaceof the skin in actual practice. Both the base array and plunger arrayscan be used in various configurations and some preferred embodiments arediscussed below.

Design 4

Use of Microimplant Cassette/Cartridge

In this modification, the drug/cell loaded microimplant is loaded orcast inside a separate cartridge (separate from base and plunger array)which may be manufactured, sterilized and packaged separately than AIAdevice or it may be pre-inserted, pre-aligned and deployed along withAIA device. The cartridge has base plate with holes/cavities whoseinternal diameter shape is same or slightly less than the holes in thebase array. The array arrangement (5 by 5 as an example is same as basearray). Holes in the cartridge are partially or completely filled withprefabricated drug/cell based unibody microimplants. The cartridge isplaced on top of base array plate with center of holes in cartridge arealigned the holes on base array plate. The plunger array needles arethen aligned with cartridge implant center, pushed. The plunger arraypushes the cartridge microimplants via base cavity needle cavity intotissue. Once in the skin/tissue, the base array, cartridge and plungerarray are removed leaving behind the implants in the cartridge in theskin/tissue.

The cartridge has a base plate with holes to store drug/cell implantsand optionally a protective covering on top and bottom plate to preventmovement of implant during storage and handling. The part of theprotective covering may be made from water soluble or biodegradablebiocompatible polymer. A stainless steel rectangular plate 500 micronsthick and 20 mm length and 20 mm width is used. At the center of theplate 25 holes with 0.29 mm dia are drilled in the 5 by 5 matrix format.The matrix position and format is same as base array needle format asshown in design 2 above. The plate is kept on Teflon sheet and the holesof the plate are filled with PEG based macromonomer solution withphotoinitiator (Example 10) and bupivacaine loaded PLGA microspheres (20percent loading). The monomer solution/suspension is exposed to long UVlight (360 nm). The polymerization forms biodegradable hydrogels insidethe holes with drug loaded microspheres. The water in the hydrogel isremoved by lyophilization. A protective polyester sheet is placed at thebottom of cartridge plate. The plate with solid lyophilizedmicroimplants with diameter 0.29 diameter is placed on top of base arrayplate with center of holes matching the center of base array cavityholes and the protective cover is removed. The base array needles arethen inserted in the sheep skin tissue and the plunger array is kept ontop of cartridge array where center of plunger needles aligns the centerof cartridge implant/holes. The plunger array is pushed downward whichpushes the hydrogel implant via base cavity array into tissue. The basearray along with cartridge and plunger array are removed from the tissueleaving the hydrogel implant for local or systemic drug therapy. Thestainless steel plate of cartridge can have holes for alignment whichcan be used with guiding posts on the base array surface.

In another variation prefabricated cylindrical implants such as shown inFIG. 15H are used. To hold the implant in place, the base plate bottomsurface has been spray/dip coated with collagen or gelatin film foam.The implants are pushed and the coated film/foam is removed just priorto use. In another embodiment same as above, the protective foam coveris not removed but is cut/stamped out by the plunger array and istransported inside the tissue and implanted (in this case plunger arrayneedles have a cutting edge to stamp out the foam). The gelatin/collagenis safely removed by the body by biodegradation process.

In another embodiment, biodegradable polymer implant (PLGA implant) iscast from a solution in situ, solvent is removed and the microimplant isinserted in the tissue as discussed before. In another variation,stainless steel base plate in the cartridge is substituted with siliconerubber sheet with holes for guide posts for alignment.

Example 22B

In Situ Formation of Microimplant Array Using “Array in Array” (AIA)Apparatus.

Using the AIA Apparatus in a Closed Configuration

Formation of microimplant array using biodegradable polymer and drug orhydrogels with live cells. In this embodiment, gelatin gel is used as amodel for skin tissue. 10 percent gelatin solution is cast into 4 mmthick 1 inch diameter gel. The “array in array” device (AIA device) asdiscussed above is used. The base array device such as shown in FIG. 15B and top array plunger device such as shown in FIG. 15D are used. Theplunger array (PA array) needles (all 25 needles) are inserted in basearray corresponding base array cavities (such as shown in FIG. 15D). Inthis arrangement, most of the hollow cavity space in the base array isoccupied by the plunger array needles. The device such as shown in FIG.15D is inserted in gelatin gel to create 5 by 5 array holes. The gelatingel cannot be cored in the cavity space of base array needles becausethe space is occupied by the plunger array needles. Upon completeinsertion, the plunger array is removed and the space/volume created bythe removal of plunger array is filled by an injectable composition. 1 gof PLGA (50:50 lactide:glycolide, PDLG 5002) polymer and 10 mg ofcoumarin and 9 ml n-methyl pyrrolidone are mixed until completesolution. The green solution (exemplary injectable composition) isinjected using a syringe in each cavity (25 total cavities). Afterfilling the cavities, excess solution is wiped off. The cavity isexposed to 2 ml PBS solution to accelerate precipitation of the polymerin the cavities. Using a plastic rod, each precipitated microimplant isindividually pushed into gelatin gel or plunger array is used to pushall implants at once. The gelatin gel with PLGA microimplants is cutinto rectangular shape and is photographed under blue light (FIG. 15G).The 5 by 5 array of PLGA based microimplants is clearly visible ingelatin gel and is fluorescent in nature (FIG. 15G). In anothermodification of above example, PLGA solution is replaced with fibringlue precursor solution with human foreskin fibroblasts cells (HFF)(about 1 million live cells in one ml of fibrinogen and thrombinmixture). The solution is allowed to form a gel in the base arraycavities and the fibrin glue gels with live HFF cells are left in thegelatin gel. A total of 25 microimplants in 5 by 5 arrangements withfibrin glue as encapsulation matrix with live HFF cells is formed in thegelatin. The gelatin gel can be substituted with live skin tissue. Inanother variation of above example, the empty space created in basearray cavity is filled with PEG based macromonomer solution (Example 10Dand 15A) are used to form soft hydrogel based arrays with live cells.Briefly, a precursor solution of macromonomer solution along withinitiator, catalysts and cells (PEG based macromonomer, eosin asinitiator and argon laser light; see Example 15 or 20 for additionaldetails) is first loaded with cells, and filled in to hollow cavities ofbase array needles as described above and exposed to argon laser lightfor 120 seconds. The light polymerizes macromonomer solution intocrosslinked gel which entraps the cells. The array is inserted in theskin tissue and the cell loaded implant is pushed out using the plungerarray needle as described above.

In another embodiment, the base array cavities of AIA device are firstfilled with biocompatible dissolvable compounds such as frozen salinesolution or PBS solution, sodium chloride powder, melted PEG polymer andthe like. The base array is inserted in the gelatin gel and thedissolvable components is absorbed in the gel or tissue creating a spacefor cavity filling.

In another embodiment, base array is first directly inserted in theporcine skin tissue and the cavities later filled injectablecompositions such as biodegradable polymer based solution (PLGA solutionwith coumarin as an example).

The precipitated polymer is left in the skin tissue after removal ofbase array needles.

In another embodiment, 1 ml of gelatin solution (5 percent in PBS,warmed to 60 degree C., exemplary thermosensitive precursor solution)and 100 mg of rifampin loaded microspheres are mixed and the warmsolution is filled in the hollow array cavities as described above. Thearray is kept in refrigerator (4 degree C. for 12 h) to form gelatin gelinside the hollow cavities. The gelatin gel with entrapped rifampinloaded PLGA microspheres is pushed out in the skin tissue using theplunger array as above. The microspheres in the implanted gelatinhydrogel in the skin tissue release rifampin in a sustained manner.

Example 22C

Formation of microimplant array using “array in array” (AIA) apparatus.

Use of prefabricated implants loaded in AIA apparatus and thenimplanted.

The base array cavities of AIA apparatus can be filled withprefabricated microimplants. In one embodiment, cut gut basedfibers/threads are first coated with PLGA and coumarin basedcompositions as described before to obtain a coated fiber. The coatedfiber is then cut into several microcylindrical rods. Average length ofcut microcylinder is 496 microns and PLGA coating had a thickness ofapproximately 40 microns (FIG. 15H). The cut microcylindrical rods areplaced inside the cavity of base array and plunger array is placed ontop of base array with 2 mm polyethylene sheet as a spacer. The deviceis transported on top of sheep skin and bottom ray needles are insertedcompletely in the skin. The spacer sheet is removed and the plungerarray is pushed in the base array cavities. The plunger array pushes theimplant in the skin. Both base array and top array are removed from theskin tissue leaving 25 implanted rods in an array format. The implantedarray can provide sustained drug delivery and fluorescent coating helpsto visualize the implants in the skin. FIG. 15H shows coatedmicrocylindrical rods in the skin tissue imaged under blue light. Theimage shows 5 by 5 matrix type implantation arrangement in the skintissue with fluorescent green coating. In some cases, a pressurizedsaline is used to push the implant from the base array cavities in thetissue. The sterile saline solution is attached to 22 gauge needle and0.5-10 psi pressurized saline is discharged from the 22 gauge needle inthe base array cavity to dislodge the implant from the array intotissue.

In another variation, synthetic crosslinked biodegradable hydrogels(microcylindrical rods with drug encapsulated microspheres are cast asmicrocylindrical rods. The drug loaded hydrogels rods are placed insidethe cavities of the AIA device and the injected in the skin tissue usingplunger array as described above. 200 mg of PEG 35K-lactate-acrylatemacromonomer (prepared per Example 10D) is dissolved in 800 mg PBS.After complete dissolution, 300 mg Irgacure 2959 is dissolved in 700 mgn-methyl pyrrolidone. 5 microliters of Irgacure 2959 solution is addedto the macromonomer solution. The solution is sterile filtered using 0.2micron filter. The sterile solution (precursor solution) mixed with 200mg of PLGA microspheres (size 10-300 microns, 10 percent dexamethasonerelative to PLGA plus drug weight) is added. The solution is filled withsilicone rubber mold with cylindrical mold cavity (size 50 microndiameter, 30 micron height) or silicone base MPatch™ Microneedletemplates from Micropoint Technologies Pte Ltd. (Singapore). The infusedsolution used then exposed to the long UV ultraviolet light (Black-RayUV lamp, 360 nm light, 10000 mW/cm2 intensity) for 5 minutes tophotopolymerize and crosslink the infused precursor solution. Thepolymerized hydrogel implants are lyophilized and are removed from themold and added in microneedle cavities of base array of AIA device asabove. The AIA base array device with implants in the cavity is infusedin the skin tissue and the implants are pushed using plunger array orpressurized nitrogen gas or pressurized saline fluid. A 22 gauge needleconnected to 0.2 micron filtered nitrogen or carbon dioxide gas cylinderand gas flow from needle is used to push each individual implant in thetissue. The base array is removed from the tissue leaving behindbiodegradable hydrogel with encapsulated hydrogel implant array in theskin tissue.

In another variation, the gelatin or PLGA solution is cast first outsideto form microimplants. The size of the implant is chosen in such amanner that they can fit inside in the cavities of hollow array. Theexternally made implants are added in the cavities of hollow array. Thearray is then inserted in the skin tissue and the implants are pushedout using epoxy based plunger array as above. In this embodiment, thearray microimplant is made first externally and then used in the deviceto implant it inside the body. The implant does not need sharp edges tobe inserted in body. The metal hollow array does the function ofpiercing the skin tissue and carrying the implant in tis cavity. Thearray plunger helps to push out the implant from the hollow array.

In another variation, 200 mg of rifampin loaded microspheres are filledin the cavities as a dry powder and then the powder ispushed/distributed in the tissue using the plunger array as describedabove. In some cases, the powder may be compacted using water solublebiocompatible and biodegradable binder like dextran or polyethyleneglycol to make a unibody implant.

Example 23

Delivery of Lyophilized Protein Drug in Solid State

Implanted Array Comprising Botulinum Toxin

Botox® is a trade name for Botulinum toxin based drug formulation. Botoxis neurotoxic protein produced by the bacterium Clostridium and is soldto treat variety of medical conditions. Botox is sold as a drylyophilized protein powder with each vial containing 50 or 100 units ofdrug and each vial contains one nanograms of drug along with salt andother additives. One Botox vial containing 100 units is diluted withsterile 0.1 ml of 0.1 percent sodium hyaluronate (mechanical propertyenhancer and bulking agent) in PBS buffer and 0.1 mg sodium fluorscein(as a coloring/fluorescent agent). The 3.53 microliter of the Botoxsolution is loaded 100 cavities of base array of 10 by 10 AIA apparatusas discussed in Example 22. The apparatus has 100 cylindrical cavitieswith 500 microns height and 310 microns diameter. The solution islyophilized in the cavities at −5-10 degree C. for 72 h. A 10 by 10plunger array of the AIA apparatus is applied on top of the base arrayaligning its needle but not inserted. A polyethylene 2 mm spacer is keptbetween the bottom and top array to prevent accidental insertion ofplunger array in the base array. Each 100 array needle containapproximately 3.53 units of Botulinum toxin. The device is terminallysterilized and used on the skin tissue. During usage, the base arrayneedles are inserted in the tissue, spacer is removed and the plungerarray is pushed to inject the fluorescent implant in the tissue. Onceinjected, both the arrays are taken out leaving behind the implant inthe tissue. Under blue light, implantation of the injected microimplantarray is visible as green array. Multiple arrays may be used to treatmore areas on the skin if needed. The sodium hyaluronate and fluorsceindissolve away leaving behind Botulinum toxin for therapeutic effect.This method delivers Botulinum toxin in solid state without dilution insaline as used in current practice.

In another embodiment, the Botulinum toxin is mixed with precursors ofcrosslinkers such as fibrin sealant and PEG-lactate macromonomersolution and crosslinked to produce biodegradable hydrogel implant withencapsulated Botulinum toxin. The encapsulated implant is then deliveredusing AIA apparatus as before. The Botulinum toxin is released in asustained manner for therapeutic action from the crosslinkedbiodegradable hydrogel.

Other protein drugs such as vaccines can be delivered in the solid stateusing similarly to Botox as described before. It is preferred that suchmicroimplants are colored or fluorescent for easy visualization ofimplanted therapeutic array. Bulking agents such as collagen, gelatin,polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, hydroxypropyl cellulose, hydroxy ethyl cellulose, dextran, maltose, chitosan,human or bovine albumin, may be added to improve mechanical handling andease of delivery. The concentration of each additive must be determinedsuch that it gives sufficient strength and bulkiness to the implantduring implantation. Generally, the concentration of additive is about0.5 to 99 percent, typically 1 to 95 percent of the total lyophilizedmass of the drug. The additive should not deactivate (not reduce oreliminate its biological activity) the Botox or other protein drug.Human serum albumin is the most preferred bulking agent for Botox.

In another modification, one hundred (200 micron diameter and 500 mmlength) lyophilized collagen sponge microcylindrical implants areobtained from commercial source (puntal implants used for dry eyetreatment). Alternatively, the 200 micron diameter implants can bestamped out from 500 micron thick collagen foam sponge. Botox solution(100 units) is diluted with 0.1 ml saline solution and each collagenimplant is treated with 1 microliter of solution of Botox solution andthe implant is immediately frozen and lyophilized. The lyophilizedimplant containing one unit of Botox drug is implanted in the skintissue using AIA array (5 by 5 stainless steel array FIGS. 15A-15H)apparatus, total dose 25 units per array per treatment. Each implantfrom the AIA apparatus cavity may be individually inserted manually inthe skin giving a dose of 1 unit of Botox per implant. The hollowmicroneedle array with preloaded microimplants as above may besterilized and packaged. The package also contains a sterile metal rodto push the implant during implantation or array of rods to push allimplants at once. This device and its method of implantation does notrequire the drug delivery implant to have sharp needle at distal endwhich allows more choice of materials to be used for sustained drugdelivery.

Example 24

Use of Live Cells for Cosmetic or Facelift Application.

Deposition of live cells using oscillating needle for faceliftapplication.

A skin sample from a human donor is used as a source material. The skinsample is carefully treated with collagenease enzyme to dissolveextracellular matrix and the live fibroblast cells are isolated from thesample. The cells are passaged 1-20 times to expand the number of cells.The expanded fibroblast cells are isolated in a sterile manner, combinedfrom all culture flasks to make a cell suspension containing 1000 to 10million live cells in 1-2 ml cell culture medium. Hyaluronic acid isadded in the medium as non-toxic a thickening agent. The suspension isinjected at in the facial tissue or other desired area using oscillatingneedle device as described before. The suspension is added avisualization agent such as fluorescent dye like sodium fluorscein orfluorescent microspheres to aid in deposition. The cells are injectedpreferably in the same donor's tissue (autologous cell therapy) at therate of 1 to 10000, preferably 1 to 5000, even more preferably 1 to 1000cells per injection. The injected live cells grow and produceextracellular matrix providing face lift effect. The distribution ofcells in the desired area, ability to cover large area in a small periodof treatment time and ability to visualize the treated area usingfluorescence of the agent provides most effective face living/cosmetictreatment.

Example 25

Drug Delivery Array Prepared Using Biodegradable Metal

Magnesium Alloy Based Drug Delivery Array

A 100 mm length, 100 mm width and 500 microns thick magnesium alloy(AZ31) foil is procured from Goodfellow Corporation, Coraopolis, Pa.,USA (product code 343-198-08). The sheet is stamped/cut to produce 5 mmlong, 500 microns wide and 500 microns thick strips. One end of thestrip is ground and sharpened to have a fine cutting edge via machining(sharp edge length 50 microns). The rest of the area is used to createholes in the body of the implant (creation of artificial porosity, laserdrilling, holes diameter 150 microns, 500 microns height, 6 holes in 2by 3 matrix format). The drilled area is polished to smoothen the edges.9 such strips are prepared. The strips with holes are dip coated in PLGAsolution containing rifampin as a model drug. 0.8 g of PLGA [(50:50lactide:glycolide, PDLG 5002)] polymer, 200 mg of rifampin and 1 mltetrahydrofuran are mixed until complete solution. The coated redsolution is dried in air and then under the vacuum. The red polymer inthe holes and on the surface of the magnesium strips is clearly visibleto the necked eye. The length of the needle is then reduced from 5 mm to1000 micron with sharp edge at distal end and middle area of the 1000long implant is occupied with 150 microns size holes filled with PLGApolymer with rifampin. 9 such implants are manually attached in 3 by 3array format and attached to the medical adhesive tape (base side ofimplant is attached to the adhesive layer of the tape and other end(distal end) has sharp edge. The needle is attached perpendicular to thetape surface. Each microneedle is separated by 2 mm on the adhesivetape, has a distal sharp end and is loaded with rifampin in PLGA asbiodegradable polymer. The array thus prepared as above is similar (butnot the same) in design as shown in FIG. 26E. The array is pressed on anexcised porcine skin tissue (sharp edge touching perpendicular to theskin surface) and the needles are inserted in the tissue. A gentlepressure is applied on the backside of the tape. The tape is gentlyremoved from the skin surface leaving behind the magnesium basedmicroimplant array in the tissue. The magnesium alloy provides abiodegradable metal based skeletal material that assists in skinpenetration. The biodegradable polymer (PLGA) provides sustained releasefunction to the array. The artificial porosity provides higher surfacearea for increased drug loading. The entire device is degradable andneed not be removed once implanted.

In another modification of above embodiment, a microneedle array withhollow metal microneedle is created and the hollow cavities of theneedle are filled with drug delivery compositions (FIG. 26A). A 300microns ID and 100 microns wall thickness magnesium AZ31 alloy tube iscut at 5 mm length. One end of the tube is cut at an angle 30 degree toproduce a sharp cutting edge. The tube cavity is then filled with PLGAsolution with rifampin in dioxane (20 percent PLGA concentration indioxane, 20 percent rifampin weight relative to PLGA weight). Thesolvent dioxane is removed lyophilization at −5 to −10 degree C. forseveral days. The tube is then cut to 1 mm in length with sharp edge atdistal end. The hollow cavity of the tube is filled with lyophilizedPLGA polymer with rifampin as a model drug. 16 such needles are preparedand are then attached to the back of adhesive tape. The base of themicroneedles is attached to the adhesive layer of the tape and distalsharp end is on the other end for skin penetration. The array is formedin 4 by 4 format with distance between the needle is at 1 mm. The arrayis implanted in the porcine skin tissue as discussed above and theadhesive tape is removed. The implanted array delivers the drug via PLGAin the needle cavity. The metal based sharp edge that enables easypenetration in the skin and the metal and PLGA based composition isremoved from the tissue by biodegradation process. Rifampin is releasedfrom the PLGA in a sustained manner.

In another modification of the above embodiment, the AZ31 foil as aboveis stamped/cut to produce 500 microns diameter and 500 microns height(sheet thickness) microcylinders. The microcylinders are then dip coatedwith rifampin solution 3 times to produce about 50 micron thick coatinglayer. A coating layer without the drug as a controlled release layer isapplied as outermost layer. The PLGA coated microcylinders are insertedin the cavity of the base array of AIA the apparatus described in FIG.6. The coated microcylinders are then implanted using the plunger arrayapparatus as described before. This microimplant array has a magnesiumbased biodegradable metal as a reinforcing agent which enables smoothpushing of drug delivery composition in the tissue. In anothermodification, a microneedle array with hybrid needle such as shown inFIG. 26C is prepared. The needle has metal based tip (2606) and drugdelivery body (cylindrical body, 2607, in this illustrative case). Theneedle only uses metal based tip (2606) for tissue penetration and drugdelivery implant body (2607) is made using biodegradable polymer orhydrogel. The body 2607 may comprise drug or live cells. The body (2607)may also be a pre-fabricated microimplant with drug or cells asdiscussed in this invention. The implant body 2607 may be porous and theporosity may be filled with drugs or cells. This design enablesimplantation of soft materials like hydrogel material with live cells.In another modification, a cage with a cavity (cavity for 2607 implantstorage in this cage) and solid tip such as shown in 2606 is made andthe prefabricated implant body (2607) is kept inside the cage cavity.The metal cage provides more rigidity to the microneedle structure andthus enables to insert materials like hydrogels to be implanted withlittle force and the entire implantation process is tolerated by thecells maintaining their viability for therapeutic use.

Although the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the appended claims. Therefore,the present embodiments are to be considered as illustrative and notrestrictive and the invention is not to be limited to the writtendescription.

Example 26

Preparation of Porous Microimplants.

Preparation Porous Synthetic Biodegradable Polymer Microimplant.

Preparation of porous structure by salt leaching.

Example 26A

Preparation of Porous Poly(L-Lactide-Co-Caprolactone Copolymer

In 250 ml beaker, 1 g of Poly(L-lactide-co-caprolactone) copolymer(70/30; PURASORB PLC 7015 from PURAC Biochem, Netherlands) is dissolvedin 10 ml 1,4-dioxane. After complete dissolution, 3 g sodium chloride(finely grounded and sieved, 75 to 125 micron particle size fraction) isadded to the mixture and the suspension is vigorously stirred. Thestirred liquid suspension is quickly frozen using liquid nitrogen. Thedioxane is removed by lyophilization. Using a 1 mm diameter cookiecutter, 1 mm cylindrical pieces are cut from the freeze-dried polymer.The cut pieces are incubated in distilled water to 24-72 h to removesodium chloride. The leaching of sodium chloride produces porousmicroimplants. Suspension could also be first poured into glass orpolyethylene tube (1 mm dia) or silicone rubber tube, frozen andlyophilized to produce a 1 mm dia freeze-dried solid. The freeze-driedsolid is cut into 100-2000 micron size sections and the sections areexposed to water for sufficient time (1-7 days) to extract sodiumchloride substantially or almost completely or completely. By changingthe particle size of porogen (sodium chloride) and its volume in theabove example, porous solids with different amount of porosity and aswell pore size can be obtained. Alternatively, the polymer solution indioxane is filled in cavities of MPatch™ silicone rubber mold cavities,centrifuged to remove air bubble and uniform filling of cavities andthen cooled in liquid nitrogen and then lyophilized. The lyophilizedporous biodegradable microneedles are separated from the mold and can beused as prefabricated porous microimplants which can be implanted usingthe AIA like device. The microneedles array needles can also be appliedan adhesive tape to make a microneedle array and porous microneedles canthen be implanted as PLGA based porous microneedle array.

Example 26B

Preparation of porous structure by leaching out organic solvent solublepolymer.

In a 250 ml conical flask, 4 g Poly (PLGA, lactide-co-glycolide)(lactide:glycolide (50:50), molecular weight 30000 to 60000 g/mole.)from Aldrich and 4 g polyethylene glycol (PEG, molecular weight 10000g/mole.) is dissolved in 100 ml warm dry dioxane under nitrogenatmosphere (50 degree C.). About 10 ml of the solution in filled in atest tube and the tube is frozen using liquid nitrogen. The frozensolution is lyophilized (freeze-dried) at −5 degree C. for 4-5 days tofreeze-dry all the solvent (dioxane). The freeze-dried polymer isremoved from the tube and is then incubated in 1000 ml beaker containing500 ml ethanol for 7-10 days. Ethanol is replaced twice a day. The PEG,which is used as an organic solvent soluble porogen is leached out inethanol and a porous PLGA polymer is obtained. The PLGA is further driedin vacuum at 50 degree C. for 3 days to remove trace amount of dioxaneand ethanol.

Example 26C

Preparation of Porous Structure by Leaching Out Organic Soluble Polymer.

Preparation of Porous Hydrophilic Biodegradable Polymer (Collagen).

In a 250 ml beaker, 100 mg Type I collagen is dissolved in 10 ml 0.5 Macetic acid. To this solution, 10 mg of poly(ethyl methacrylate)spherical beads are added (from Polysciences, Particle size 140-220microns,) are added. The suspension is stirred and quickly frozen usingliquid nitrogen. The frozen solution is lyophilized to remove water andthen transferred 100 ml methanol. The poly(ethyl methacrylate) isextracted out in methanol for 7 days with fresh methanol exchanged everyday. The dissolution of poly(ethyl methacrylate) by methanol createsporosity in the collagen matrix. In a similar embodiment, 10 g Poly(L-lactide) PURASORB PL 18 is completely dissolved in 100 ml dioxane. 5g poly(ethyl methacrylate) microspheres are added to the mixture and themixture is quickly frozen before solvent corrodes the microspheres andthen freeze dried at −5 degree C. The freeze-dried product is thensubjected to extraction with methanol wherein only microspheres aresoluble and Poly (L-lactide) is not soluble. The extracted microspheresleave behind spherical empty space or cavities or porosity which can beused to fill other degradable polymer with drugs.

Example 26D

Porosity using mechanical or laser drilling or injection molding.

A high molecular weight polycaprolactone or polylactic acid cylindricalrod (1 mm diameter) is extruded or injection molded from a laboratorybased plastic processing extrusion machine or injection molding machine.Alternatively the rod can be solution casted in a cylindrical mold andsolvent is removed. The extruded/injection rod is cut to 0.5 mm dia and0.5 mm length and the pieces are subjected to laser drilling ormechanical drilling. Several 25-100 microns in diameter and 50-100microns in depth cylindrical holes are drilled on the polymer rodsurface. UV, infrared or visible light based laser systems are used todrill the holes.

Example 26E

Porous structures can be made by knitting and/or weaving biodegradablefibers. Fibers suitable for implantation may be knitted or weaved tocreate a fabric with certain amount of porosity. The weaving andknitting pattern may be changed to create small or large amount of emptyspace between fibers to obtain a desirable porous volume. Additionally,fabrics may be used as a starting material to create cavities in thefabric structure.

Example 27

Delivery of in situ forming crosslinkable compositions can be performedby using oscillating needle devices. This can can include delivery of acomposition that crosslinks via an enzymatic pathway. The formation offibrin gel particles can be performed in situ.

A commercially available EVICEL® from Ethicon or TISSEEL® from Baxtermay be used. The components of fibrin glue (fibrinogen, thrombin, factor8, calcium ions and the like) are supplied as a two component mixture.The components of commercially available fibrin sealant are mixed in asterile cup (total volume of mixed components 1-2 ml). To this solution5 drops ophthalmic sodium fluorscein solution are added or 10 mg ofindocyanine green dye added as a fluorescent/coloring agent or coloringagent. If no color is desired, the formulation can be used without theuse of coloring agent or dye. The colored fibrin formulation is thenloaded in the oscillating needle of the tattoo machine and injected(tattooed) into pericardial tissue or in live skin tissue. The excesssolution on the tissue surface is wiped off. The injected solutiondroplets undergo enzymatic polymerization/crosslinking and form fibringlue/gel particles in situ inside the tissue. Care is taken to injectthe formulation before the fibrin glue forms gel (usually 1-2 minutes).If components prematurely gel, then a new mixture is prepared and usedquickly. The fibrinogen solution may be diluted using PBS to slow thegelation process. Alternatively a modified tattoo machine like device isused wherein the components are mixed inside the device just prior toinjection and injected by an oscillating needle. The colorant orfluorescence of particles or droplets provides visual clue on the amountof injected solution at each injection site. A drug may be added to thecomposition. Drugs that interfere with the fibrin glue formation such asTPA or heparin cannot be used for local delivery using this method. Manydrugs can be used with fibrin glue system. Live cell suspensions may beadded to the fibrin glue components to deliver live cell basedcompositions. A multilumen needle may be used to deliver fibrin glueprecursors (one lumen for fibrinogen solution) and another lumen forthrombin solution. The components are injected simultaneously andcrosslinked in situ.

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The invention claimed is:
 1. A method of making a sustainedbiodegradable drug delivery implant in a live tissue, the methodcomprising: creating at least two artificial cavities of predeterminedshape and volume in the live tissue; filling the at least two artificialcavities partially or completely with an injectable biodegradablecomposition comprising a biodegradable polymer dissolved in abiocompatible, water-soluble organic solvent; and precipitating thepolymer from the injected polymer solution so as to form the sustainedbiodegradable implant in the artificial cavity.
 2. The method of claim1, further comprising forming at least 4 artificial cavities per squarecentimeter in the live tissue.
 3. The method of claim 1, furthercomprising forming from 4 artificial cavities to 6,000 artificialcavities per square centimeter in the live tissue.
 4. The method ofclaim 1, wherein a volume of each cavity may range from about 1×10E-12ml to about 0.05 ml.
 5. The method of claim 3, further comprisingforming an array of the artificial cavities in the live tissue.
 6. Themethod of claim 3, further comprising forming the cavities to have aspacing between adjacent cavities to range from 1 micron to 10 mm. 7.The method of claim 3, wherein the creating of the artificial cavititesfurther comprises displacing tissue, destroying tissue, or combinationthereof.
 8. The method of claim 1, wherein the creating of theartificial cavitites further comprises laser ablation, microneedlearray, oscillating needle, mechanical drilling, ultrasonic drilling,water drilling, or combination thereof.
 9. The method of claim 1,wherein the tissue is skin tissue.
 10. The method of claim 1, whereinthe biocompatible, water-soluble organic solvent is selected from agroup consisting of tripropionin (triprop), tetraglycol, pyrrolidone-2,ethyl lactate, triacetin, triethylene glycol dimethyl ether (triglyme),glycerol formal, dimethyl sulfoxide, ethylene glycol monoethyl etheracetate, benzyl alcohol, n-methyl pyrrolidone, N-ethyl-2-pyrrolidone,tributyrin, benzyl benzoate, acetone, methyl ethyl ketone, acetic acid,ethanol, isopropanol, diethylene glycol dimethyl ether (Diglyme), ethylbenzoate, dimethyl isosorbide (DMI), polyethylene glycol dimethyl ether,glycofurol, glycerol, ethyl acetate, polyethylene glycol (low molecularweight), 1,3 propane diol, 1,4 butane diol, 1-6-hexane diol,tetrahydrofuran, triethanol amine, water, buffered water solutions withpH ranging from 6 to 8, and combinations thereof.
 11. The method ofclaim 1, wherein a concentration of the biocompatible polymer in thebiocompatible, water-soluble organic solvent ranges from about 0.1% toabout 60%.
 12. The method of claim 1, wherein the injectablebiodegradable composition and formed implant comprises a visualizationagent.
 13. The method of claim 12, wherein the visualization agent is acolored compound, a fluorescent compound, an x-ray imaging agent, radioopaque contrast agent, NMR contrast agent, or a MRI agent.
 14. Themethod of claim 1, comprising injecting the injectable biodegradablecomposition into the tissue at a depth of about 10 microns to about 5mm.
 15. The method of claim 1, wherein the injectable biodegradablecomposition and formed implant comprises a drug.
 16. The method of claim15, wherein the drug is dissolved, suspended, or emulsified in theinjectable composition before the filing of the at least two cavitites.17. The method of claim 16, wherein the drug is present at a drugconcentration of about 1% to 40% relative to polymer weight of thebiodegradable polymer in polymer solution or formed implant.
 18. Themethod of claim 15, wherein the drug is selected from the groupconsisting of: antiinfectives, antibiotics; antiviral agents, antifungalagents, antibacterial agents, antipruritics; anticancer agents,antipsychotics; cholesterol- or lipid-reducing agents; cell cycleinhibitors; anticancer agents; antiparkinsonism drugs; HMG-CoAinhibitors; antirestenosis agents; antiinflammatory agents;antiasthmatic agents; anthelmintic; immunosuppressives; musclerelaxants; antidiuretic agents; vasodilators; nitric oxide; nitricoxide-releasing compounds; beta-blockers; hormones; antidepressants;decongestants; calcium channel blockers; growth factors, bone growthfactors or bone morphogenic proteins; wound healing agents; analgesicsand analgesic combinations; local anesthetic agents; antihistamines;sedatives; angiogenesis-promoting agents; angiogenesis-inhibitingagents; tranquilizers; and combinations thereof.
 19. The method of claim1, further comprising forming the at least two cavities to have a depthof about 0.3 microns to about 3.5 mm.
 20. The method of claim 1, furthercomprising injecting the injectable biodegradable composition into thetissue at an amount of 1.0E-02 ml to 1.0E-16 ml per injection.